BASB013 DNA and proteins from neisseria meningitidis

ABSTRACT

The invention provides BASB013 polypeptides and polynucleotides encoding BASB013 polypeptides and methods for producing such polypeptides by recombinant techniques. Also provided are diagnostic, prophylactic and therapeutic uses.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. patentapplication Ser. No. 09/673,898 filed Dec. 4, 2000, which is theNational Stage Application of International Application No.PCT/EP99/02765, filed Apr. 20, 1999 which was published under PCTarticle 21(2) in English, which claims the benefit of priority of GreatBritain Patent Application Serial No. 9808734.9, filed Apr. 23, 1998.The disclosure of these applications are hereby incorporated byreference as if fully set forth herein.

FIELD OF THE INVENTION

[0002] This invention relates to polynucleotides, (herein referred to as“BASB013 polynucleotide(s)”), polypeptides encoded by them (referred toherein as “BASB013” or “BASB013 polypeptide(s)”), recombinant materialsand methods for their production. In another aspect, the inventionrelates to methods for using such polypeptides and polynucleotides,including vaccines against bacterial infections. In a further aspect,the invention relates to diagnostic assays for detecting infection ofcertain pathogens.

BACKGROUND OF THE INVENTION

[0003]Neisseria meningitidis (meningococcus) is a Gram negativebacterium frequently isolated from the human upper respiratory tract. Itoccasionally causes invasive bacterial diseases such as bacteremia andmeningitis. The incidence of meningococcal disease shows geographicalseasonal and annual differences (Schwartz, B., Moore, P. S., Broome, C.V.; Clin. Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Most diseasein temperate countries is due to strains of serogroup B and varies inincidence from 1-10/100,000/year total population sometimes reachinghigher values (Kaczmarski, E. B. (1997), Commun. Dis. Rep. Rev. 7:R55-9, 1995; Scholten, R. J. P. M., Bijlmer, H. A., Poolman, J. T. etal. Clin. Infect. Dis. 16: 237-246, 1993; Cruz, C., Pavez, G., Aguilar,E., et al. Epidemiol. Infect. 105: 119-126, 1990).

[0004] Epidemics dominated by serogroup A meningococci, mostly incentral Africa, are encountered, sometimes reaching levels up to1000/100.000/year (Schwartz, B., Moore, P. S., Broome, C. V. Clin.Microbiol. Rev. 2 (Supplement), S18-S24, 1989). Nearly all cases as awhole of meningococcal disease are caused by serogroup A, B, C, W-135and Y meningococci and a tetravalent A, C, W-135, Y polysaccharidevaccine is available (Armand, J., Arminjon, F., Mynard, M. C., Lafaix,C., J. Biol. Stand. 10: 335-339, 1982).

[0005] The polysaccharide vaccines are currently being improved by wayof chemical conjugating them to carrier proteins (Lieberman, J. M.,Chiu, S. S., Wong, V. K., et al. JAMA 275: 1499-1503,1996).

[0006] A serogroup B vaccine is not available, since the B capsularpolysaccharide was found to be nonimmunogenic, most likely because itshares structural similarity to host components (Wyle, F. A.,Artenstein, M. S., Brandt, M. L. et al. J. Infect. Dis. 126: 514-522,1972; Finne, J. M., Leinonen, M., Mäkelä, P. M. Lancet ii.: 355-357,1983).

[0007] For many years efforts have been initiated and carried out todevelop meningococcal outer membrane based vaccines (de Moraes, J. C.,Perkins, B., Camargo, M. C. et al. Lancet 340: 1074-1078, 1992; Bjune,G., Hoiby, E. A. Gronnesby, J. K. et al. 338: 1093-1096, 1991). Suchvaccines have demonstrated efficcacies from 57%-85% in older children(>4 years) and adolescents.

[0008] Many bacterial outer membrane components are present in thesevaccines, such as PorA, PorB, Rmp, Opc, Opa, FrpB and the contributionof these components to the observed protection still needs futherdefinition. Other bacterial outer membrane components have been definedby using animal or human antibodies to be potentially relevant to theinduction of protective immunity, such as TbpB and NspA (Martin, D.,Cadieux, N., Hamel, J., Brodeux, B. R., J. Exp. Med. 185: 1173-1183,1997; Lissolo, L., Maître-Wilmotte, C., Dumas, p. et al., Inf. Immun.63: 884-890, 1995). The mechanisms of protective immunity will involveantibody mediated bactericidal activity and opsonophagocytosis.

[0009] A bacteremia animal model has been used to combine all antibodymediated mechanisms (Saukkonen, K., Leinonen, M., Abdillahi, H. Poolman,J. T. Vaccine 7: 325-328, 1989). It is generally accepted that the latecomplement component mediated bactericidal mechanism is crucial forimmunity against meningococcal disease (Ross, S. C., Rosenthal P. J.,Berberic, H. M., Densen, P. J. Infect. Dis. 155: 1266-1275, 1987).

[0010] The frequency of Neisseria meningitidis infections has risendramatically in the past few decades. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Neisseria meningitidis strains that are resistant tosome or all of the standard antibiotics. This phenomenon has created anunmet medical need and demand for new anti-microbial agents, vaccines,drug screening methods, and diagnostic tests for this organism.

SUMMARY OF THE INVENTION

[0011] The present invention relates to BASB013, in particular BASB013polypeptides and BASB013 polynucleotides, recombinant materials andmethods for their production. In another aspect, the invention relatesto methods for using such polypeptides and polynucleotides, includingprevention and treatment of microbial diseases, amongst others. In afurther aspect, the invention relates to diagnostic assays for detectingdiseases associated with microbial infections and conditions associatedwith such infections, such as assays for detecting expression oractivity of BASB013 polynucleotides or polypeptides.

[0012] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following descriptions and from reading theother parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS. 1A-1E show consecutive alignments for three BASB013polynucleotide sequences.

[0014] FIGS. 2A-2B show consecutive alignments for three BASB013polypeptide sequences.

[0015]FIG. 3 shows substantially purified, recombinant BASB013 formsseparated on SDS-PAGE, and stained with Coomassie Blue.

[0016]FIG. 4 shows anti-BASB013 antibodies in human convalescent sera(part B) and mice immunized with Outer Membrane Proteins of H44/76Neisseria meningitidis cells (part A).

DESCRIPTION OF THE INVENTION

[0017] The invention relates to BASB013 polypeptides and polynucleotidesas described in greater detail below. In particular, the inventionrelates to polypeptides and polynucleotides of BASB013 of Neisseriameningitidis, which is related by amino acid sequence homology to theMucD protein of Pseudomonas aeruginosa. The invention relates especiallyto BASB013 having the nucleotide and amino acid sequences set out in SEQID NO: 1, 3, 5 and SEQ ID NO:2, 4, 6 respectively. It is understood thatsequences recited in the Sequence Listing below as “DNA” represent anexemplification of one embodiment of the invention, since those ofordinary skill will recognize that such sequences can be usefullyemployed in polynucleotides in general, including ribopolynucleotides.

[0018] Polypeptides

[0019] In one aspect of the invention there are provided polypeptides ofNeisseria meningitidis referred to herein as “BASB013” and “BASB013polypeptides” as well as biologically, diagnostically, prophylactically,clinically or therapeutically useful variants thereof, and compositionscomprising the same.

[0020] The present invention further provides for:

[0021] (a) an isolated polypeptide which comprises an amino acidsequence which has at least 85% identity, more preferably at least 90%identity, yet more preferably at least 95% identity, most preferably atleast 97-99% or exact identity, to that of SEQ ID NO:2, 4, 6;

[0022] (b) a polypeptide encoded by an isolated polynucleotidecomprising a polynucleotide sequence which has at least 85% identity,more preferably at least 90% identity, yet more preferably at least 95%identity, even more preferably at least 97-99% or exact identity to SEQID NO: 1, 3, 5 over the entire length of SEQ ID NO: 1, 3, 5respectively; or

[0023] (c) a polypeptide encoded by an isolated polynucleotidecomprising a polynucleotide sequence encoding a polypeptide which has atleast 85% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%or exact identity, to the amino acid sequence of SEQ ID NO:2, 4, 6;

[0024] The BASB013 polypeptides provided in SEQ ID NO:2, 4, 6 are theBASB013 polypeptides from Neisseria meningitidis strains ATCC 13090 andH44/76.

[0025] The invention also provides an immunogenic fragment of a BASB013polypeptide, that is, a contiguous portion of the BASB013 polypeptidewhich has the same or substantially the same immunogenic activity as thepolypeptide comprising the amino acid sequence of SEQ ID NO:2, 4, 6.That is to say, the fragment (if necessary when coupled to a carrier) iscapable of raising an immune response which recognises the BASB013polypeptide. Such an immunogenic fragment may include, for example, theBASB013 polypeptide lacking an N-terminal leader sequence, and/or atransmembrane domain and/or a C-terminal anchor domain. In a preferredaspect the immunogenic fragment of BASB013 according to the inventioncomprises substantially all of the extracellular domain of a polypeptidewhich has at least 85% identity, more preferably at least 90% identity,yet more preferably at least 95% identity, most preferably at least97-99% identity, to that of SEQ ID NO:2, 4, 6 over the entire length ofSEQ ID NO:4.

[0026] A fragment is a polypeptide having an amino acid sequence that isentirely the same as part but not all of any amino acid sequence of anypolypeptide of the invention. As with BASB013 polypeptides, fragmentsmay be “free-standing,” or comprised within a larger polypeptide ofwhich they form a part or region, most preferably as a single continuousregion in a single larger polypeptide.

[0027] Preferred fragments include, for example, truncation polypeptideshaving a portion of an amino acid sequence of SEQ ID NO:2, 4, 6 or ofvariants thereof, such as a continuous series of residues that includesan amino- and/or carboxyl-terminal amino acid sequence. Degradationforms of the polypeptides of the invention produced by or in a hostcell, are also preferred. Further preferred are fragments characterizedby structural or functional attributes such as fragments that comprisealpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions.

[0028] Further preferred fragments include an isolated polypeptidecomprising an amino acid sequence having at least 15, 20, 30, 40, 50 or100 contiguous amino acids from the amino acid sequence of SEQ ID NO:2,4, 6 or an isolated polypeptide comprising an amino acid sequence havingat least 15, 20, 30, 40, 50 or 100 contiguous amino acids truncated ordeleted from the amino acid sequence of SEQ ID NO:2, 4, 6.

[0029] Fragments of the polypeptides of the invention may be employedfor producing the corresponding full-length polypeptide by peptidesynthesis; therefore, these fragments may be employed as intermediatesfor producing the full-length polypeptides of the invention.

[0030] Particularly preferred are variants in which several, 5-10,1-5,1-3, 1-2 or 1 amino acids are substituted, deleted, or added in anycombination.

[0031] The polypeptides, or immunogenic fragments, of the invention maybe in the form of the “mature” protein or may be a part of a largerprotein such as a precursor or a fusion protein. It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification such as multiple histidine residues, or an additionalsequence for stability during recombinant production. Furthermore,addition of exogenous polypeptide or lipid tail or polynucleotidesequences to increase the immunogenic potential of the final molecule isalso considered.

[0032] In one aspect, the invention relates to genetically engineeredsoluble fusion proteins comprising a polypeptide of the presentinvention, or a fragment thereof, and various portions of the constantregions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa.

[0033] Furthermore, this invention relates to processes for thepreparation of these fusion proteins by genetic engineering, and to theuse thereof for drug screening, diagnosis and therapy. A further aspectof the invention also relates to polynucleotides encoding such fusionproteins. Examples of fusion protein technology can be found inInternational Patent Application Nos. WO94/29458 and WO94/22914.

[0034] The proteins may be chemically conjugated, or expressed asrecombinant fusion proteins allowing increased levels to be produced inan expression system as compared to non-fused protein. The fusionpartner may assist in providing T helper epitopes (immunological fusionpartner), preferably T helper epitopes recognised by humans, or assistin expressing the protein (expression enhancer) at higher yields thanthe native recombinant protein. Preferably the fusion partner will beboth an immunological fusion partner and expression enhancing partner.

[0035] Fusion partners include protein D from Haemophilus influenzae andthe non-structural protein from influenzae virus, NS1 (hemagglutinin).Another fusion partner is the protein known as LytA. Preferably the Cterminal portion of the molecule is used. LytA is derived fromStreptococcus pneumoniae which synthesize an N-acetyl-L-alanine amidase,amidase LytA, (coded by the lytA gene {Gene, 43 (1986) page 265-272}) anautolysin that specifically degrades certain bonds in the peptidoglycanbackbone. The C-terminal domain of the LytA protein is responsible forthe affinity to the choline or to some choline analogues such as DEAE.This property has been exploited for the development of E. coli C-LytAexpressing plasmids useful for expression of fusion proteins.Purification of hybrid proteins containing the C-LytA fragment at itsamino terminus has been described {Biotechnology: 10, (1992) page795-798}. It is possible to use the repeat portion of the LytA moleculefound in the C terminal end starting at residue 178, for exampleresidues 188-305.

[0036] The present invention also includes variants of theaforementioned polypeptides, that is polypeptides that vary from thereferents by conservative amino acid substitutions, whereby a residue issubstituted by another with like characteristics. Typical suchsubstitutions are among Ala, Val, Leu and Ile; among Ser and Thr; amongthe acidic residues Asp and Glu; among Asn and Gln; and among the basicresidues Lys and Arg; or aromatic residues Phe and Tyr.

[0037] Polypeptides of the present invention can be prepared in anysuitable manner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

[0038] It is most preferred that a polypeptide of the invention isderived from Neisseria meningitidis, however, it may preferably beobtained from other organisms of the same taxonomic genus. A polypeptideof the invention may also be obtained, for example, from organisms ofthe same taxonomic family or order.

[0039] Polynucleotides

[0040] It is an object of the invention to provide polynucleotides thatencode BASB013 polypeptides, particularly polynucleotides that encodethe polypeptide herein designated BASB013.

[0041] In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding BASB013 polypeptidescomprising a sequence set out in SEQ ID NO:1, 3, 5 which includes a fulllength gene, or a variant thereof.

[0042] The BASB013 polynucleotides provided in SEQ ID NO:1, 3, 5 are theBASB013 polynucleotides from Neisseria meningitidis strains ATCC 13090and H44/76.

[0043] As a further aspect of the invention there are provided isolatednucleic acid molecules encoding and/or expressing BASB013 polypeptidesand polynucleotides, particularly Neisseria meningitidis BASB013polypeptides and polynucleotides, including, for example, unprocessedRNAs, ribozyme RNAs, mRNAs, cDNAs, genomic DNAs, B- and Z-DNAs. Furtherembodiments of the invention include biologically, diagnostically,prophylactically, clinically or therapeutically useful polynucleotidesand polypeptides, and variants thereof, and compositions comprising thesame.

[0044] Another aspect of the invention relates to isolatedpolynucleotides, including at least one full length gene, that encodes aBASB013 polypeptide having a deduced amino acid sequence of SEQ ID NO:2,4, 6 and polynucleotides closely related thereto and variants thereof.

[0045] In another particularly preferred embodiment of the inventionthere is a BASB013 polypeptide from Neisseria meningitidis comprising orconsisting of an amino acid sequence of SEQ ID NO:2, 4, 6 or a variantthereof.

[0046] Using the information provided herein, such as a polynucleotidesequence set out in SEQ ID NO: 1, 3, 5, a polynucleotide of theinvention encoding BASB013 polypeptide may be obtained using standardcloning and screening methods, such as those for cloning and sequencingchromosomal DNA fragments from bacteria using Neisseria meningitidiscells as starting material, followed by obtaining a full length clone.For example, to obtain a polynucleotide sequence of the invention, suchas a polynucleotide sequence given in SEQ ID NO: 1, 3, 5, typically alibrary of clones of chromosomal DNA of Neisseria meningitidis in E.coli or some other suitable host is probed with a radiolabeledoligonucleotide, preferably a 17-mer or longer, derived from a partialsequence. Clones carrying DNA identical to that of the probe can then bedistinguished using stringent hybridization conditions. By sequencingthe individual clones thus identified by hybridization with sequencingprimers designed from the original polypeptide or polynucleotidesequence it is then possible to extend the polynucleotide sequence inboth directions to determine a full length gene sequence. Conveniently,such sequencing is performed, for example, using denatured doublestranded DNA prepared from a plasmid clone. Suitable techniques aredescribed by Maniatis, T., Fritsch, E. F. and Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989). (see in particular Screening ByHybridization 1.90 and Sequencing Denatured Double-Stranded DNATemplates 13.70). Direct genomic DNA sequencing may also be performed toobtain a full length gene sequence. Illustrative of the invention, eachpolynucleotide set out in SEQ ID NO: 1, 3, 5 was discovered in a DNAlibrary derived from Neisseria meningitidis

[0047] Moreover, each DNA sequence set out in SEQ ID NO: 1, 3, 5contains an open reading frame encoding a protein having about thenumber of amino acid residues set forth in SEQ ID NO:2, 4, 6 with adeduced molecular weight that can be calculated using amino acid residuemolecular weight values well known to those skilled in the art.

[0048] The polynucleotide of SEQ ID NO:1, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1498 of SEQ ID NO: 1, encodes the polypeptide of SEQ ID NO:2.

[0049] The polynucleotide of SEQ ID NO:3, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1498 of SEQ ID NO:3, encodes the polypeptide of SEQ ID NO:4.

[0050] The polynucleotide of SEQ ID NO:5, between the start codon atnucleotide number 1 and the stop codon which begins at nucleotide number1498 of SEQ ID NO:5, encodes the polypeptide of SEQ ID NO:6.

[0051] In a further aspect, the present invention provides for anisolated polynucleotide comprising or consisting of:

[0052] (a) a polynucleotide sequence which has at least 85% identity,more preferably at least 90% identity, yet more preferably at least 95%identity, even more preferably at least 97-99% or exact identity to SEQID NO: 1, 3, 5 over the entire length of SEQ ID NO:1, 3, 5 respectively;or

[0053] (b) a polynucleotide sequence encoding a polypeptide which has atleast 85% identity, more preferably at least 90% identity, yet morepreferably at least 95% identity, even more preferably at least 97-99%or 100% exact, to the amino acid sequence of SEQ ID NO:2, 4, 6 over theentire length of SEQ ID NO:2, 4, 6 respectively.

[0054] A polynucleotide encoding a polypeptide of the present invention,including homologs and orthologs from species other than Neisseriameningitidis, may be obtained by a process which comprises the steps ofscreening an appropriate library under stringent hybridizationconditions (for example, using a temperature in the range of 45-65° C.and an SDS concentration from 0.1-1%) with a labeled or detectable probeconsisting of or comprising the sequence of SEQ ID NO: 1, 3, 5 or afragment thereof; and isolating a full-length gene and/or genomic clonescontaining said polynucleotide sequence.

[0055] The invention provides a polynucleotide sequence identical overits entire length to a coding sequence (open reading frame) in SEQ IDNO: 1, 3, 5. Also provided by the invention is a coding sequence for amature polypeptide or a fragment thereof, by itself as well as a codingsequence for a mature polypeptide or a fragment in reading frame withanother coding sequence, such as a sequence encoding a leader orsecretory sequence, a pre-, or pro- or prepro-protein sequence. Thepolynucleotide of the invention may also contain at least one non-codingsequence, including for example, but not limited to at least onenon-coding 5′ and 3′ sequence, such as the transcribed butnon-translated sequences, termination signals (such as rho-dependent andrho-independent termination signals), ribosome binding sites, Kozaksequences, sequences that stabilize mRNA, introns, and polyadenylationsignals. The polynucleotide sequence may also comprise additional codingsequence encoding additional amino acids. For example, a marker sequencethat facilitates purification of the fused polypeptide can be encoded.In certain embodiments of the invention, the marker sequence is ahexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) anddescribed in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821-824(1989), or an HA peptide tag (Wilson et al., Cell 37: 767 (1984), bothof which may be useful in purifying polypeptide sequence fused to them.Polynucleotides of the invention also include, but are not limited to,polynucleotides comprising a structural gene and its naturallyassociated sequences that control gene expression.

[0056] The nucleotide sequence encoding BASB013 polypeptide of SEQ IDNO:2, 4, 6 may be identical to the polypeptide encoding sequencecontained in nucleotides 1 to 1497 of SEQ ID NO: 1, or the polypeptideencoding sequence contained in nucleotides 1 to 1497 of SEQ ID NO:3; orthe polypeptide encoding sequence contained in nucleotides 1 to 1497 ofSEQ ID NO:5, respectively. Alternatively it may be a sequence, which asa result of the redundancy (degeneracy) of the genetic code, alsoencodes the polypeptide of SEQ ID NO:2, 4, 6.

[0057] The term “polynucleotide encoding a polypeptide” as used hereinencompasses polynucleotides that include a sequence encoding apolypeptide of the invention, particularly a bacterial polypeptide andmore particularly a polypeptide of the Neisseria meningitidis BASB013having an amino acid sequence set out in SEQ ID NO:2, 4, 6. The termalso encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,polynucleotides interrupted by integrated phage, an integrated insertionsequence, an integrated vector sequence, an integrated transposonsequence, or due to RNA editing or genomic DNA reorganization) togetherwith additional regions, that also may contain coding and/or non-codingsequences.

[0058] The invention further relates to variants of the polynucleotidesdescribed herein that encode variants of a polypeptide having a deducedamino acid sequence of SEQ ID NO:2, 4, 6. Fragments of polynucleotidesof the invention may be used, for example, to synthesize full-lengthpolynucleotides of the invention.

[0059] Further particularly preferred embodiments are polynucleotidesencoding BASB013 variants, that have the amino acid sequence of BASB013polypeptide of SEQ ID NO:2, 4, 6 in which several, a few, 5 to 10, 1 to5, 1 to 3, 2, 1 or no amino acid residues are substituted, modified,deleted and/or added, in any combination. Especially preferred amongthese are silent substitutions, additions and deletions, that do notalter the properties and activities of BASB013 polypeptide.

[0060] Further preferred embodiments of the invention arepolynucleotides that are at least 85% identical over their entire lengthto a polynucleotide encoding BASB013 polypeptide having an amino acidsequence set out in SEQ ID NO:2, 4, 6, and polynucleotides that arecomplementary to such polynucleotides. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

[0061] Preferred embodiments are polynucleotides encoding polypeptidesthat retain substantially the same biological function or activity asthe mature polypeptide encoded by a DNA of SEQ ID NO: 1, 3, 5.

[0062] In accordance with certain preferred embodiments of thisinvention there are provided polynucleotides that hybridize,particularly under stringent conditions, to BASB013 polynucleotidesequences, such as those polynucleotides in SEQ ID NO: 1, 3, 5.

[0063] The invention further relates to polynucleotides that hybridizeto the polynucleotide sequences provided herein. In this regard, theinvention especially relates to polynucleotides that hybridize understringent conditions to the polynucleotides described herein. As hereinused, the terms “stringent conditions” and “stringent hybridizationconditions” mean hybridization occurring only if there is at least 95%and preferably at least 97% identity between the sequences. A specificexample of stringent hybridization conditions is overnight incubation at42° C. in a solution comprising: 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml of denatured,sheared salmon sperm DNA, followed by washing the hybridization supportin 0.1×SSC at about 65° C. Hybridization and wash conditions are wellknown and exemplified in Sambrook, et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989),particularly Chapter 11 therein. Solution hybridization may also be usedwith the polynucleotide sequences provided by the invention.

[0064] The invention also provides a polynucleotide consisting of orcomprising a polynucleotide sequence obtained by screening anappropriate library containing the complete gene for a polynucleotidesequence set forth in SEQ ID NO: 1, 3, 5 under stringent hybridizationconditions with a probe having the sequence of said polynucleotidesequence set forth in SEQ ID NO: 1, 3, 5 or a fragment thereof; andisolating said polynucleotide sequence. Fragments useful for obtainingsuch a polynucleotide include, for example, probes and primers fullydescribed elsewhere herein.

[0065] As discussed elsewhere herein regarding polynucleotide assays ofthe invention, for instance, the polynucleotides of the invention, maybe used as a hybridization probe for RNA, cDNA and genomic DNA toisolate full-length cDNAs and genomic clones encoding BASB013 and toisolate cDNA and genomic clones of other genes that have a highidentity, particularly high sequence identity, to the BASB013 gene. Suchprobes generally will comprise at least 15 nucleotide residues or basepairs. Preferably, such probes will have at least 30 nucleotide residuesor base pairs and may have at least 50 nucleotide residues or basepairs. Particularly preferred probes will have at least 20 nucleotideresidues or base pairs and will have less than 30 nucleotide residues orbase pairs.

[0066] A coding region of a BASB013 gene may be isolated by screeningusing a DNA sequence provided in SEQ ID NO: 1, 3, 5 to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the invention is then used to screena library of cDNA, genomic DNA or mRNA to determine which members of thelibrary the probe hybridizes to.

[0067] There are several methods available and well known to thoseskilled in the art to obtain full-length DNAs, or extend short DNAs, forexample those based on the method of Rapid Amplification of cDNA ends(RACE) (see, for example, Frohman, et al., PNAS USA 85: 8998-9002,1988). Recent modifications of the technique, exemplified by theMarathon™ technology (Clontech Laboratories Inc.) for example, havesignificantly simplified the search for longer cDNAs. In the Marathon™technology, cDNAs have been prepared from mRNA extracted from a chosentissue and an ‘adaptor’ sequence ligated onto each end. Nucleic acidamplification (PCR) is then carried out to amplify the “missing” 5′ endof the DNA using a combination of gene specific and adaptor specificoligonucleotide primers. The PCR reaction is then repeated using“nested” primers, that is, primers designed to anneal within theamplified product (typically an adaptor specific primer that annealsfurther 3′ in the adaptor sequence and a gene specific primer thatanneals further 5′ in the selected gene sequence). The products of thisreaction can then be analyzed by DNA sequencing and a full-length DNAconstructed either by joining the product directly to the existing DNAto give a complete sequence, or carrying out a separate full-length PCRusing the new sequence information for the design of the 5′ primer.

[0068] The polynucleotides and polypeptides of the invention may beemployed, for example, as research reagents and materials for discoveryof treatments of and diagnostics for diseases, particularly humandiseases, as further discussed herein relating to polynucleotide assays.

[0069] The polynucleotides of the invention that are oligonucleotidesderived from a sequence of SEQ ID NOS: 1-6 may be used in the processesherein as described, but preferably for PCR, to determine whether or notthe polynucleotides identified herein in whole or in part aretranscribed in bacteria in infected tissue. It is recognized that suchsequences will also have utility in diagnosis of the stage of infectionand type of infection the pathogen has attained.

[0070] The invention also provides polynucleotides that encode apolypeptide that is the mature protein plus additional amino orcarboxyl-terminal amino acids, or amino acids interior to the maturepolypeptide (when the mature form has more than one polypeptide chain,for instance). Such sequences may play a role in processing of a proteinfrom precursor to a mature form, may allow protein transport, maylengthen or shorten protein half-life or may facilitate manipulation ofa protein for assay or production, among other things. As generally isthe case in vivo, the additional amino acids may be processed away fromthe mature protein by cellular enzymes.

[0071] For each and every polynucleotide of the invention there isprovided a polynucleotide complementary to it. It is preferred thatthese complementary polynucleotides are fully complementary to eachpolynucleotide with which they are complementary.

[0072] A precursor protein, having a mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0073] In addition to the standard A, G, C, T/U representations fornucleotides, the term “N” may also be used in describing certainpolynucleotides of the invention. “N” means that any of the four DNA orRNA nucleotides may appear at such a designated position in the DNA orRNA sequence, except it is preferred that N is not a nucleic acid thatwhen taken in combination with adjacent nucleotide positions, when readin the correct reading frame, would have the effect of generating apremature termination codon in such reading frame.

[0074] In sum, a polynucleotide of the invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences that are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

[0075] In accordance with an aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization.

[0076] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet (1992) 1: 363, Manthorpe et al., Hum. Gene Ther. (1983) 4: 419),delivery of DNA complexed with specific protein carriers (Wu et al., JBiol. Chem. (1989) 264: 16985), coprecipitation of DNA with calciumphosphate (Benvenisty & Reshef, PNAS USA, (1986) 83: 9551),encapsulation of DNA in various forms of liposomes (Kaneda et al.,Science (1989) 243: 375), particle bombardment (Tang et al., Nature(1992) 356:152, Eisenbraun et al., DNA Cell Biol (1993) 12: 791) and invivo infection using cloned retroviral vectors (Seeger et al., PNAS USA(1984) 81: 5849).

[0077] Vectors, Host Cells, Expression Systems

[0078] The invention also relates to vectors that comprise apolynucleotide or polynucleotides of the invention, host cells that aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the invention.

[0079] Recombinant polypeptides of the present invention may be preparedby processes well known in those skilled in the art from geneticallyengineered host cells comprising expression systems. Accordingly, in afurther aspect, the present invention relates to expression systems thatcomprise a polynucleotide or polynucleotides of the present invention,to host cells which are genetically engineered with such expressionsystems, and to the production of polypeptides of the invention byrecombinant techniques.

[0080] For recombinant production of the polypeptides of the invention,host cells can be genetically engineered to incorporate expressionsystems or portions thereof or polynucleotides of the invention.Introduction of a polynucleotide into the host cell can be effected bymethods described in many standard laboratory manuals, such as Davis, etal., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989), such as, calciumphosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introductionand infection.

[0081] Representative examples of appropriate hosts include bacterialcells, such as cells of streptococci, staphylococci, enterococci, E.coli, streptomyces, cyanobacteria, Bacillus subtilis, Moraxellacatarrhalis, Haemophilus influenzae and Neisseria meningitidis; fungalcells, such as cells of a yeast, Kluveromyces, Saccharomyces, abasidiomycete, Candida albicans and Aspergillus; insect cells such ascells of Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,COS, HeLa, C127, 3T3, BHK, 293, CV-1 and Bowes melanoma cells; and plantcells, such as cells of a gymnosperm or angiosperm.

[0082] A great variety of expression systems can be used to produce thepolypeptides of the invention. Such vectors include, among others,chromosomal-, episomal- and virus-derived vectors, for example, vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses, picomaviruses, retroviruses, and alphaviruses and vectorsderived from combinations thereof, such as those derived from plasmidand bacteriophage genetic elements, such as cosmids and phagemids. Theexpression system constructs may contain control regions that regulateas well as engender expression. Generally, any system or vector suitableto maintain, propagate or express polynucleotides and/or to express apolypeptide in a host may be used for expression in this regard. Theappropriate DNA sequence may be inserted into the expression system byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, (supra).

[0083] In recombinant expression systems in eukaryotes, for secretion ofa translated protein into the lumen of the endoplasmic reticulum, intothe periplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0084] Polypeptides of the present invention can be recovered andpurified from recombinant cell cultures by well-known methods includingammonium sulfate or ethanol precipitation, acid extraction, anion orcation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography and lectin chromatography. Mostpreferably, ion metal affinity chromatography (IMAC) is employed forpurification. Well known techniques for refolding proteins may beemployed to regenerate active conformation when the polypeptide isdenatured during intracellular synthesis, isolation and or purification.

[0085] The expression system may also be a recombinant livemicroorganism, such as a virus or bacterium. The gene of interest can beinserted into the genome of a live recombinant virus or bacterium.Inoculation and in vivo infection with this live vector will lead to invivo expression of the antigen and induction of immune responses.Viruses and bacteria used for this purpose are for instance: poxviruses(e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus,Semliki Forest Virus, Venezuelian Equine Encephalitis Virus),adenoviruses, adeno-associated virus, picomaviruses (poliovirus,rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria,Salmonella, Shigella, Neisseria, BCG. These viruses and bacteria can bevirulent, or attenuated in various ways in order to obtain livevaccines. Such live vaccines also form part of the invention.

[0086] Diagnostic, Prognostic, Serotyping and Mutation Assays

[0087] This invention is also related to the use of BASB013polynucleotides and polypeptides of the invention for use as diagnosticreagents. Detection of BASB013 polynucleotides and/or polypeptides in aeukaryote, particularly a mammal, and especially a human, will provide adiagnostic method for diagnosis of disease, staging of disease orresponse of an infectious organism to drugs. Eukaryotes, particularlymammals, and especially humans, particularly those infected or suspectedto be infected with an organism comprising the BASB013 gene or protein,may be detected at the nucleic acid or amino acid level by a variety ofwell known techniques as well as by methods provided herein.

[0088] Polypeptides and polynucleotides for prognosis, diagnosis orother analysis may be obtained from a putatively infected and/orinfected individual's bodily materials. Polynucleotides from any ofthese sources, particularly DNA or RNA, may be used directly fordetection or may be amplified enzymatically by using PCR or any otheramplification technique prior to analysis. RNA, particularly mRNA, cDNAand genomic DNA may also be used in the same ways. Using amplification,characterization of the species and strain of infectious or residentorganism present in an individual, may be made by an analysis of thegenotype of a selected polynucleotide of the organism. Deletions andinsertions can be detected by a change in size of the amplified productin comparison to a genotype of a reference sequence selected from arelated organism, preferably a different species of the same genus or adifferent strain of the same species. Point mutations can be identifiedby hybridizing amplified DNA to labeled BASB013 polynucleotidesequences. Perfectly or significantly matched sequences can bedistinguished from imperfectly or more significantly mismatched duplexesby DNase or RNase digestion, for DNA or RNA respectively, or bydetecting differences in melting temperatures or renaturation kinetics.Polynucleotide sequence differences may also be detected by alterationsin the electrophoretic mobility of polynucleotide fragments in gels ascompared to a reference sequence. This may be carried out with orwithout denaturing agents. Polynucleotide differences may also bedetected by direct DNA or RNA sequencing. See, for example, Myers etal., Science, 230: 1242 (1985). Sequence changes at specific locationsalso may be revealed by nuclease protection assays, such as RNase, V1and S1 protection assay or a chemical cleavage method. See, for example,Cotton et al., Proc. Natl. Acad. Sci., USA, 85: 4397-4401 (1985).

[0089] In another embodiment, an array of oligonucleotides probescomprising BASB013 nucleotide sequence or fragments thereof can beconstructed to conduct efficient screening of, for example, geneticmutations, serotype, taxonomic classification or identification. Arraytechnology methods are well known and have general applicability and canbe used to address a variety of questions in molecular geneticsincluding gene expression, genetic linkage, and genetic variability(see, for example, Chee et al., Science, 274: 610 (1996)).

[0090] Thus in another aspect, the present invention relates to adiagnostic kit which comprises:

[0091] (a) a polynucleotide of the present invention, preferably thenucleotide sequence of SEQ ID NO: 1, 3, 5 or a fragment thereof;

[0092] (b) a nucleotide sequence complementary to that of (a);

[0093] (c) a polypeptide of the present invention, preferably thepolypeptide of SEQ ID NO:2, 4, 6 or a fragment thereof; or

[0094] (d) an antibody to a polypeptide of the present invention,preferably to the polypeptide of SEQ ID NO:2, 4, 6.

[0095] It will be appreciated that in any such kit, (a), (b), (c) or (d)may comprise a substantial component. Such a kit will be of use indiagnosing a disease or susceptibility to a disease, among others.

[0096] This invention also relates to the use of polynucleotides of thepresent invention as diagnostic reagents. Detection of a mutated form ofa polynucleotide of the invention, preferable, SEQ ID NO:1, 3, 5 whichis associated with a disease or pathogenicity will provide a diagnostictool that can add to, or define, a diagnosis of a disease, a prognosisof a course of disease, a determination of a stage of disease, or asusceptibility to a disease, which results from under-expression,over-expression or altered expression of the polynucleotide. Organisms,particularly infectious organisms, carrying mutations in suchpolynucleotide may be detected at the polynucleotide level by a varietyof techniques, such as those described elsewhere herein.

[0097] Cells from an organism carrying mutations or polymorphisms(allelic variations) in a polynucleotide and/or polypeptide of theinvention may also be detected at the polynucleotide or polypeptidelevel by a variety of techniques, to allow for serotyping, for example.For example, RT-PCR can be used to detect mutations in the RNA. It isparticularly preferred to use RT-PCR in conjunction with automateddetection systems, such as, for example, GeneScan. RNA, cDNA or genomicDNA may also be used for the same purpose, PCR. As an example, PCRprimers complementary to a polynucleotide encoding BASB013 polypeptidecan be used to identify and analyze mutations.

[0098] The invention further provides primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying BASB013 DNA and/or RNA isolatedfrom a sample derived from an individual, such as a bodily material. Theprimers may be used to amplify a polynucleotide isolated from aninfected individual, such that the polynucleotide may then be subject tovarious techniques for elucidation of the polynucleotide sequence. Inthis way, mutations in the polynucleotide sequence may be detected andused to diagnose and/or prognose the infection or its stage or course,or to serotype and/or classify the infectious agent.

[0099] The invention further provides a process for diagnosing disease,preferably bacterial infections, more preferably infections caused byNeisseria meningitidis, comprising determining from a sample derivedfrom an individual, such as a bodily material, an increased level ofexpression of polynucleotide having a sequence of SEQ ID NO: 1, 3, 5.Increased or decreased expression of a BASB013 polynucleotide can bemeasured using any on of the methods well known in the art for thequantitation of polynucleotides, such as, for example, amplification,PCR, RT-PCR, RNase protection, Northern blotting, spectrometry and otherhybridization methods.

[0100] In addition, a diagnostic assay in accordance with the inventionfor detecting over-expression of BASB013 polypeptide compared to normalcontrol tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a BASB013 polypeptide, in a sample derived from a host, suchas a bodily material, are well-known to those of skill in the art. Suchassay methods include radioimmunoassays, competitive-binding assays,Western Blot analysis, antibody sandwich assays, antibody detection andELISA assays.

[0101] The polynucleotides of the invention may be used as components ofpolynucleotide arrays, preferably high density arrays or grids. Thesehigh density arrays are particularly useful for diagnostic andprognostic purposes. For example, a set of spots each comprising adifferent gene, and further comprising a polynucleotide orpolynucleotides of the invention, may be used for probing, such as usinghybridization or nucleic acid amplification, using a probe obtained orderived from a bodily sample, to determine the presence of a particularpolynucleotide sequence or related sequence in an individual. Such apresence may indicate the presence of a pathogen, particularly Neisseriameningitidis, and may be useful in diagnosing and/or prognosing diseaseor a course of disease. A grid comprising a number of variants of thepolynucleotide sequence of SEQ ID NO: 1, 3, 5 are preferred. Alsopreferred is a grid comprising a number of variants of a polynucleotidesequence encoding the polypeptide sequence of SEQ ID NO:2, 4, 6.

[0102] Antibodies

[0103] The polypeptides and polynucleotides of the invention or variantsthereof, or cells expressing the same can be used as immunogens toproduce antibodies immunospecific for such polypeptides orpolynucleotides respectively.

[0104] In certain preferred embodiments of the invention there areprovided antibodies against BASB013 polypeptides or polynucleotides.

[0105] Antibodies generated against the polypeptides or polynucleotidesof the invention can be obtained by administering the polypeptidesand/or polynucleotides of the invention, or epitope-bearing fragments ofeither or both, analogues of either or both, or cells expressing eitheror both, to an animal, preferably a nonhuman, using routine protocols.For preparation of monoclonal antibodies, any technique known in the artthat provides antibodies produced by continuous cell line cultures canbe used. Examples include various techniques, such as those in Kohler,G. and Milstein, C., Nature 256. 495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0106] Techniques for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies topolypeptides or polynucleotides of this invention. Also, transgenicmice, or other organisms or animals, such as other mammals, may be usedto express humanized antibodies immunospecific to the polypeptides orpolynucleotides of the invention.

[0107] Alternatively, phage display technology may be utilized to selectantibody genes with binding activities towards a polypeptide of theinvention either from repertoires of PCR amplified v-genes oflymphocytes from humans screened for possessing anti-BASB013 or fromnaive libraries (McCafferty, et al., (1990), Nature 348, 552-554; Marks,et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by, for example, chain shuffling(Clackson et al., (1991) Nature 352: 628).

[0108] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptides or polynucleotides of theinvention to purify the polypeptides or polynucleotides by, for example,affinity chromatography.

[0109] Thus, among others, antibodies against BASB013-polypeptide orBASB013-polynucleotide may be employed to treat infections, particularlybacterial infections.

[0110] Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants form a particular aspect of thisinvention.

[0111] Preferably, the antibody or variant thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanized,” where thecomplimentarity determining region or regions of the hybridoma-derivedantibody has been transplanted into a human monoclonal antibody, forexample as described in Jones et al. (1986), Nature 321, 522-525 orTempest et al., (1991) Biotechnology 9, 266-273.

[0112] Antagonists and Agonists—Assays and Molecules

[0113] Polypeptides and polynucleotides of the invention may also beused to assess the binding of small molecule substrates and ligands in,for example, cells, cell-free preparations, chemical libraries, andnatural product mixtures. These substrates and ligands may be naturalsubstrates and ligands or may be structural or functional mimetics. See,e.g., Coligan et al., Current Protocols in Immunology 1(2): Chapter 5(1991).

[0114] The screening methods may simply measure the binding of acandidate compound to the polypeptide or polynucleotide, or to cells ormembranes bearing the polypeptide or polynucleotide, or a fusion proteinof the polypeptide by means of a label directly or indirectly associatedwith the candidate compound. Alternatively, the screening method mayinvolve competition with a labeled competitor. Further, these screeningmethods may test whether the candidate compound results in a signalgenerated by activation or inhibition of the polypeptide orpolynucleotide, using detection systems appropriate to the cellscomprising the polypeptide or polynucleotide. Inhibitors of activationare generally assayed in the presence of a known agonist and the effecton activation by the agonist by the presence of the candidate compoundis observed. Constitutively active polypeptide and/or constitutivelyexpressed polypeptides and polynucleotides may be employed in screeningmethods for inverse agonists or inhibitors, in the absence of an agonistor inhibitor, by testing whether the candidate compound results ininhibition of activation of the polypeptide or polynucleotide, as thecase may be. Further, the screening methods may simply comprise thesteps of mixing a candidate compound with a solution containing apolypeptide or polynucleotide of the present invention, to form amixture, measuring BASB013 polypeptide and/or polynucleotide activity inthe mixture, and comparing the BASB013 polypeptide and/or polynucleotideactivity of the mixture to a standard. Fusion proteins, such as thosemade from Fc portion and BASB013 polypeptide, as hereinbefore described,can also be used for high-throughput screening assays to identifyantagonists of the polypeptide of the present invention, as well as ofphylogenetically and and/or functionally related polypeptides (see D.Bennett et al., J Mol Recognition, 8:52-58 (1995); and K. Johanson etal., J Biol Chem, 270(16):9459-9471 (1995)).

[0115] The polynucleotides, polypeptides and antibodies that bind toand/or interact with a polypeptide of the present invention may also beused to configure screening methods for detecting the effect of addedcompounds on the production of mRNA and/or polypeptide in cells. Forexample, an ELISA assay may be constructed for measuring secreted orcell associated levels of polypeptide using monoclonal and polyclonalantibodies by standard methods known in the art. This can be used todiscover agents which may inhibit or enhance the production ofpolypeptide (also called antagonist or agonist, respectively) fromsuitably manipulated cells or tissues.

[0116] The invention also provides a method of screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof BASB013 polypeptides or polynucleotides, particularly those compoundsthat are bacteristatic and/or bactericidal. The method of screening mayinvolve high-throughput techniques. For example, to screen for agonistsor antagonists, a synthetic reaction mix, a cellular compartment, suchas a membrane, cell envelope or cell wall, or a preparation of anythereof, comprising BASB013 polypeptide and a labeled substrate orligand of such polypeptide is incubated in the absence or the presenceof a candidate molecule that may be a BASB013 agonist or antagonist. Theability of the candidate molecule to agonize or antagonize the BASB013polypeptide is reflected in decreased binding of the labeled ligand ordecreased production of product from such substrate. Molecules that bindgratuitously, i.e., without inducing the effects of BASB013 polypeptideare most likely to be good antagonists. Molecules that bind well and, asthe case may be, increase the rate of product production from substrate,increase signal transduction, or increase chemical channel activity areagonists. Detection of the rate or level of, as the case may be,production of product from substrate, signal transduction, or chemicalchannel activity may be enhanced by using a reporter system. Reportersystems that may be useful in this regard include but are not limited tocolorimetric, labeled substrate converted into product, a reporter genethat is responsive to changes in BASB013 polynucleotide or polypeptideactivity, and binding assays known in the art.

[0117] Another example of an assay for BASB013 agonists is a competitiveassay that combines BASB013 and a potential agonist with BASB013-bindingmolecules, recombinant BASB013 binding molecules, natural substrates orligands, or substrate or ligand mimetics, under appropriate conditionsfor a competitive inhibition assay. BASB013 can be labeled, such as byradioactivity or a colorimetric compound, such that the number ofBASB013 molecules bound to a binding molecule or converted to productcan be determined accurately to assess the effectiveness of thepotential antagonist.

[0118] Potential antagonists include, among others, small organicmolecules, peptides, polypeptides and antibodies that bind to apolynucleotide and/or polypeptide of the invention and thereby inhibitor extinguish its activity or expression. Potential antagonists also maybe small organic molecules, a peptide, a polypeptide such as a closelyrelated protein or antibody that binds the same sites on a bindingmolecule, such as a binding molecule, without inducing BASB013-inducedactivities, thereby preventing the action or expression of BASB013polypeptides and/or polynucleotides by excluding BASB013 polypeptidesand/or polynucleotides from binding.

[0119] Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of BASB006.

[0120] In a further aspect, the present invention relates to geneticallyengineered soluble fusion proteins comprising a polypeptide of thepresent invention, or a fragment thereof, and various portions of theconstant regions of heavy or light chains of immunoglobulins of varioussubclasses (IgG, IgM, IgA, IgE). Preferred as an immunoglobulin is theconstant part of the heavy chain of human IgG, particularly IgG1, wherefusion takes place at the hinge region. In a particular embodiment, theFc part can be removed simply by incorporation of a cleavage sequencewhich can be cleaved with blood clotting factor Xa. Furthermore, thisinvention relates to processes for the preparation of these fusionproteins by genetic engineering, and to the use thereof for drugscreening, diagnosis and therapy. A further aspect of the invention alsorelates to polynucleotides encoding such fusion proteins. Examples offusion protein technology can be found in International PatentApplication Nos. WO94/29458 and WO94/22914.

[0121] Each of the polynucleotide sequences provided herein may be usedin the discovery and development of antibacterial compounds. The encodedprotein, upon expression, can be used as a target for the screening ofantibacterial drugs. Additionally, the polynucleotide sequences encodingthe amino terminal regions of the encoded protein or Shine-Delgarno orother translation facilitating sequences of the respective mRNA can beused to construct antisense sequences to control the expression of thecoding sequence of interest.

[0122] The invention also provides the use of the polypeptide,polynucleotide, agonist or antagonist of the invention to interfere withthe initial physical interaction between a pathogen or pathogens and aeukaryotic, preferably mammalian, host responsible for sequelae ofinfection. In particular, the molecules of the invention may be used: inthe prevention of adhesion of bacteria, in particular gram positiveand/or gram negative bacteria, to eukaryotic, preferably mammalian,extracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; to block bacterial adhesion betweeneukaryotic, preferably mammalian, extracellular matrix proteins andbacterial BASB013 proteins that mediate tissue damage and/or; to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

[0123] In accordance with yet another aspect of the invention, there areprovided BASB013 agonists and antagonists, preferably bacteristatic orbactericidal agonists and antagonists.

[0124] The antagonists and agonists of the invention may be employed,for instance, to prevent, inhibit and/or treat diseases.

[0125] In a further aspect, the present invention relates to mimotopesof the polypeptide of the invention. A mimotope is a peptide sequence,sufficiently similar to the native peptide (sequentially orstructurally), which is capable of being recognised by antibodies whichrecognise the native peptide; or is capable of raising antibodies whichrecognise the native peptide when coupled to a suitable carrier.

[0126] Peptide mimotopes may be designed for a particular purpose byaddition, deletion or substitution of elected amino acids. Thus, thepeptides may be modified for the purposes of ease of conjugation to aprotein carrier. For example, it may be desirable for some chemicalconjugation methods to include a terminal cysteine. In addition it maybe desirable for peptides conjugated to a protein carrier to include ahydrophobic terminus distal from the conjugated terminus of the peptide,such that the free unconjugated end of the peptide remains associatedwith the surface of the carrier protein. Thereby presenting the peptidein a conformation which most closely resembles that of the peptide asfound in the context of the whole native molecule. For example, thepeptides may be altered to have an N-terminal cysteine and a C-terminalhydrophobic amidated tail. Alternatively, the addition or substitutionof a D-stereoisomer form of one or more of the amino acids may beperformed to create a beneficial derivative, for example to enhancestability of the peptide.

[0127] Alternatively, peptide mimotopes may be identified usingantibodies which are capable themselves of binding to the polypeptidesof the present invention using techniques such as phage displaytechnology (EP 0 552 267 B1). This technique, generates a large numberof peptide sequences which mimic the structure of the native peptidesand are, therefore, capable of binding to anti-native peptideantibodies, but may not necessarily themselves share significantsequence homology to the native polypeptide.

[0128] Vaccines

[0129] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal,preferably humans, which comprises inoculating the individual withBASB013 polynucleotide and/or polypeptide, or a fragment or variantthereof, adequate to produce antibody and/or T cell immune response toprotect said individual from infection, particularly bacterial infectionand most particularly Neisseria meningitidis infection. Also providedare methods whereby such immunological response slows bacterialreplication. Yet another aspect of the invention relates to a method ofinducing immunological response in an individual which comprisesdelivering to such individual a nucleic acid vector, sequence orribozyme to direct expression of BASB013 polynucleotide and/orpolypeptide, or a fragment or a variant thereof, for expressing BASB013polynucleotide and/or polypeptide, or a fragment or a variant thereof invivo in order to induce an immunological response, such as, to produceantibody and/or T cell immune response, including, for example,cytokine-producing T cells or cytotoxic T cells, to protect saidindividual, preferably a human, from disease, whether that disease isalready established within the individual or not. One example ofadministering the gene is by accelerating it into the desired cells as acoating on particles or otherwise. Such nucleic acid vector may compriseDNA, RNA, a ribozyme, a modified nucleic acid, a DNA/RNA hybrid, aDNA-protein complex or an RNA-protein complex.

[0130] A further aspect of the invention relates to an immunologicalcomposition that when introduced into an individual, preferably a human,capable of having induced within it an immunological response, inducesan immunological response in such individual to a BASB013 polynucleotideand/or polypeptide encoded therefrom, wherein the composition comprisesa recombinant BASB013 polynucleotide and/or polypeptide encodedtherefrom and/or comprises DNA and/or RNA which encodes and expresses anantigen of said BASB013 polynucleotide, polypeptide encoded therefrom,or other polypeptide of the invention. The immunological response may beused therapeutically or prophylactically and may take the form ofantibody immunity and/or cellular immunity, such as cellular immunityarising from CTL or CD4+ T cells.

[0131] A BASB013 polypeptide or a fragment thereof may be fused withco-protein or chemical moiety which may or may not by itself produceantibodies, but which is capable of stabilizing the first protein andproducing a fused or modified protein which will have antigenic and/orimmunogenic properties, and preferably protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Haemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, or any otherrelatively large co-protein which solubilizes the protein andfacilitates production and purification thereof. Moreover, theco-protein may act as an adjuvant in the sense of providing ageneralized stimulation of the immune system of the organism receivingthe protein. The co-protein may be attached to either the amino- orcarboxy-terminus of the first protein.

[0132] Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides and/orpolynucleotides of the invention and immunostimulatory DNA sequences,such as those described in Sato, Y. et al. Science 273: 352 (1996).

[0133] Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof, which have been shown toencode non-variable regions of bacterial cell surface proteins, inpolynucleotide constructs used in such genetic immunization experimentsin animal models of infection with Neisseria meningitidis. Suchexperiments will be particularly useful for identifying protein epitopesable to provoke a prophylactic or therapeutic immune response. It isbelieved that this approach will allow for the subsequent preparation ofmonoclonal antibodies of particular value, derived from the requisiteorgan of the animal successfully resisting or clearing infection, forthe development of prophylactic agents or therapeutic treatments ofbacterial infection, particularly Neisseria meningitidis infection, inmammals, particularly humans.

[0134] The invention also includes a vaccine formulation which comprisesan immunogenic recombinant polypeptide and/or polynucleotide of theinvention together with a suitable carrier, such as a pharmaceuticallyacceptable carrier. Since the polypeptides and polynucleotides may bebroken down in the stomach, each is preferably administeredparenterally, including, for example, administration that issubcutaneous, intramuscular, intravenous, or intradermal. Formulationssuitable for parenteral administration include aqueous and non-aqueoussterile injection solutions which may contain anti-oxidants, buffers,bacteristatic compounds and solutes which render the formulationisotonic with the bodily fluid, preferably the blood, of the individual;and aqueous and non-aqueous sterile suspensions which may includesuspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use.

[0135] The vaccine formulation of the invention may also includeadjuvant systems for enhancing the immunogenicity of the formulation.Preferably the adjuvant system raises preferentially a TH1 type ofresponse.

[0136] An immune response may be broadly distinguished into two extremecatagories, being a humoral or cell mediated immune responses(traditionally characterised by antibody and cellular effectormechanisms of protection respectively). These categories of responsehave been termed TH1-type responses (cell-mediated response), andTH2-type immune responses (humoral response).

[0137] Extreme TH1-type immune responses may be characterised by thegeneration of antigen specific, haplotype restricted cytotoxic Tlymphocytes, and natural killer cell responses. In mice TH1-typeresponses are often characterised by the generation of antibodies of theIgG2a subtype, whilst in the human these correspond to IgG1 typeantibodies. TH2-type immune responses are characterised by thegeneration of a broad range of immunoglobulin isotypes including in miceIgG1, IgA, and IgM.

[0138] It can be considered that the driving force behind thedevelopment of these two types of immune responses are cytokines. Highlevels of TH1-type cytokines tend to favour the induction of cellmediated immune responses to the given antigen, whilst high levels ofTH2-type cytokines tend to favour the induction of humoral immuneresponses to the antigen.

[0139] The distinction of TH1 and TH2-type immune responses is notabsolute. In reality an individual will support an immune response whichis described as being predominantly TH1 or predominantly TH2. However,it is often convenient to consider the families of cytokines in terms ofthat described in murine CD4 +ve T cell clones by Mosmann and Coffman(Mosmann, T. R. and Coffman, R. L. (1989) TH1 and TH2 cells: differentpatterns of lymphokine secretion lead to different functionalproperties. Annual Review of Immunology, 7, p 145-173). Traditionally,TH1-type responses are associated with the production of the INF-γ andIL-2 cytokines by T-lymphocytes. Other cytokines often directlyassociated with the induction of TH1-type immune responses are notproduced by T-cells, such as IL-12. In contrast, TH2-type responses areassociated with the secretion of IL-4, IL-5, IL-6 and IL-13.

[0140] It is known that certain vaccine adjuvants are particularlysuited to the stimulation of either TH1 or TH2-type cytokine responses.Traditionally the best indicators of the TH1:TH2 balance of the immuneresponse after a vaccination or infection includes direct measurement ofthe production of TH1 or TH2 cytokines by T lymphocytes in vitro afterrestimulation with antigen, and/or the measurement of the IgG1:IgG2aratio of antigen specific antibody responses.

[0141] Thus, a TH1-type adjuvant is one which preferentially stimulatesisolated T-cell populations to produce high levels of TH1-type cytokineswhen re-stimulated with antigen in vitro, and promotes development ofboth CD8+cytotoxic T lymphocytes and antigen specific immunoglobulinresponses associated with TH1-type isotype.

[0142] Adjuvants which are capable of preferential stimulation of theTH1 cell response are

[0143] described in International Patent Application No. WO 94/00153 andWO 95/17209.

[0144] 3 De-O-acylated monophosphoryl lipid A (3D-MPL) is one suchadjuvant. This is known from GB 2220211 (Ribi). Chemically it is amixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6acylated chains and is manufactured by Ribi Immunochem, Montana. Apreferred form of 3 De-O-acylated monophosphoryl lipid A is disclosed inEuropean Patent 0 689 454 B1 (SmithKline Beecham Biologicals SA).

[0145] Preferably, the particles of 3D-MPL are small enough to besterile filtered through a 0.22 micron membrane (European Patent number0 689 454).

[0146] 3D-MPL will be present in the range of 10 μg-100 μg preferably25-50 μg per dose wherein the antigen will typically be present in arange 2-50 μg per dose.

[0147] Another preferred adjuvant comprises QS21, an Hplc purifiednon-toxic fraction derived from the bark of Quillaja Saponaria Molina.Optionally this may be admixed with 3 De-O-acylated monophosphoryl lipidA (3D-MPL), optionally together with a carrier.

[0148] The method of production of QS21 is disclosed in U.S. Pat. No.5,057,540.

[0149] Non-reactogenic adjuvant formulations containing QS21 have beendescribed previously (WO 96/33739). Such formulations comprising QS21and cholesterol have been shown to be successful TH1 stimulatingadjuvants when formulated together with an antigen.

[0150] Further adjuvants which are preferential stimulators of TH1 cellresponse include immunomodulatory oligonucleotides, for exampleunmethylated CpG sequences as disclosed in WO 96/02555.

[0151] Combinations of different TH1 stimulating adjuvants, such asthose mentioned hereinabove, are also contemplated as providing anadjuvant which is a preferential stimulator of TH1 cell response. Forexample, QS21 can be formulated together with 3D-MPL. The ratio of QS21:3D-MPL will typically be in the order of 1: 10 to 10: 1; preferably 1:5to 5: 1 and often substantially 1:1. The preferred range for optimalsynergy is 2.5: 1 to 1:1 3D-MPL: QS21.

[0152] Preferably a carrier is also present in the vaccine compositionaccording to the invention. The carrier may be an oil in water emulsion,or an aluminium salt, such as aluminium phosphate or aluminiumhydroxide.

[0153] A preferred oil-in-water emulsion comprises a metabolisible oil,such as squalene, alpha tocopherol and Tween 80. In a particularlypreferred aspect the antigens in the vaccine composition according tothe invention are combined with QS21 and 3D-MPL in such an emulsion.Additionally the oil in water emulsion may contain span 85 and/orlecithin and/or tricaprylin.

[0154] Typically for human administration QS21 and 3D-MPL will bepresent in a vaccine in the range of 1 μg-200 μg, such as 10-100 μg,preferably 10 μg-50 μg per dose. Typically the oil in water willcomprise from 2 to 10% squalene, from 2 to 10% alpha tocopherol and from0.3 to 3% tween 80. Preferably the ratio of squalene: alpha tocopherolis equal to or less than 1 as this provides a more stable emulsion. Span85 may also be present at a level of 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabiliser.

[0155] Non-toxic oil in water emulsions preferably contain a non-toxicoil, e.g. squalane or squalene, an emulsifier, e.g. Tween 80, in anaqueous carrier. The aqueous carrier may be, for example, phosphatebuffered saline.

[0156] A particularly potent adjuvant formulation involving QS21, 3D-MPLand tocopherol in an oil in water emulsion is described in WO 95/17210.

[0157] The present invention also provides a polyvalent vaccinecomposition comprising a vaccine formulation of the invention incombination with other antigens, in particular antigens useful fortreating cancers, autoimmune diseases and related conditions. Such apolyvalent vaccine composition may include a TH-1 inducing adjuvant ashereinbefore described.

[0158] While the invention has been described with reference to certainBASB013 polypeptides and polynucleotides, it is to be understood thatthis covers fragments of the naturally occurring polypeptides andpolynucleotides, and similar polypeptides and polynucleotides withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant polypeptides orpolynucleotides.

[0159] The antigen can also be delivered in the form of whole bacteria(dead or alive) or as subcellular fractions, these possibilities doinclude N. meningitidis itself.

[0160] Compositions, Kits and Administration

[0161] In a further aspect of the invention there are providedcompositions comprising a BASB013 polynucleotide and/or a BASB013polypeptide for administration to a cell or to a multicellular organism.

[0162] The invention also relates to compositions comprising apolynucleotide and/or a polypeptide discussed herein or their agonistsor antagonists. The polypeptides and polynucleotides of the inventionmay be employed in combination with a non-sterile or sterile carrier orcarriers for use with cells, tissues or organisms, such as apharmaceutical carrier suitable for administration to an individual.Such compositions comprise, for instance, a media additive or atherapeutically effective amount of a polypeptide and/or polynucleotideof the invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof. Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

[0163] Polypeptides, polynucleotides and other compounds of theinvention may be employed alone or in conjunction with other compounds,such as therapeutic compounds.

[0164] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0165] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0166] In a further aspect, the present invention provides forpharmaceutical compositions comprising a therapeutically effectiveamount of a polypeptide and/or polynucleotide, such as the soluble formof a polypeptide and/or polynucleotide of the present invention, agonistor antagonist peptide or small molecule compound, in combination with apharmaceutically acceptable carrier or excipient. Such carriers include,but are not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The invention furtherrelates to pharmaceutical packs and kits comprising one or morecontainers filled with one or more of the ingredients of theaforementioned compositions of the invention. Polypeptides,polynucleotides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0167] The composition will be adapted to the route of administration,for instance by a systemic or an oral route. Preferred forms of systemicadministration include injection, typically by intravenous injection.Other injection routes, such as subcutaneous, intramuscular, orintraperitoneal, can be used. Alternative means for systemicadministration include transmucosal and transdermal administration usingpenetrants such as bile salts or fusidic acids or other detergents. Inaddition, if a polypeptide or other compounds of the present inventioncan be formulated in an enteric or an encapsulated formulation, oraladministration may also be possible. Administration of these compoundsmay also be topical and/or localized, in the form of salves, pastes,gels, solutions, powders and the like.

[0168] For administration to mammals, and particularly humans, it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0169] The dosage range required depends on the choice of peptide, theroute of administration, the nature of the formulation, the nature ofthe subject's condition, and the judgment of the attending practitioner.Suitable dosages, however, are in the range of 0.1-100 μg/kg of subject.

[0170] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response. Asuitable unit dose for vaccination is 0.5-5 microgram/kg of antigen, andsuch dose is preferably administered 1-3 times and with an interval of1-3 weeks. With the indicated dose range, no adverse toxicologicaleffects will be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0171] Wide variations in the needed dosage, however, are to be expectedin view of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Variations in these dosagelevels can be adjusted using standard empirical routines foroptimization, as is well understood in the art.

[0172] Sequence Databases, Sequences in a Tangible Medium, andAlgorithms

[0173] Polynucleotide and polypeptide sequences form a valuableinformation resource with which to determine their 2- and 3-dimensionalstructures as well as to identify further sequences of similar homology.These approaches are most easily facilitated by storing the sequence ina computer readable medium and then using the stored data in a knownmacromolecular structure program or to search a sequence database usingwell known searching tools, such as the GCG program package.

[0174] Also provided by the invention are methods for the analysis ofcharacter sequences or strings, particularly genetic sequences orencoded protein sequences. Preferred methods of sequence analysisinclude, for example, methods of sequence homology analysis, such asidentity and similarity analysis, DNA, RNA and protein structureanalysis, sequence assembly, cladistic analysis, sequence motifanalysis, open reading frame determination, nucleic acid base calling,codon usage analysis, nucleic acid base trimming, and sequencingchromatogram peak analysis.

[0175] A computer based method is provided for performing homologyidentification. This method comprises the steps of: providing a firstpolynucleotide sequence comprising the sequence of a polynucleotide ofthe invention in a computer readable medium; and comparing said firstpolynucleotide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

[0176] A computer based method is also provided for performing homologyidentification, said method comprising the steps of: providing a firstpolypeptide sequence comprising the sequence of a polypeptide of theinvention in a computer readable medium; and comparing said firstpolypeptide sequence to at least one second polynucleotide orpolypeptide sequence to identify homology.

[0177] All publications and references, including but not limited topatents and patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

[0178] DEFINITIONS

[0179] “Identity,” as known in the art, is a relationship between two ormore polypeptide sequences or two or more polynucleotide sequences, asthe case may be, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”can be readily calculated by known methods, including but not limited tothose described in (Computational Molecular Biology, Lesk, A. M., ed.,Oxford University Press, New York, 1988; Biocomputing. Informatics andGenome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heine, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math.,48: 1073 (1988). Methods to determine identity are designed to give thelargest match between the sequences tested. Moreover, methods todetermine identity are codified in publicly available computer programs.Computer program methods to determine identity between two sequencesinclude, but are not limited to, the GAP program in the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), and FASTA (Pearson and Lipman Proc. Natl. Acad. Sci. USA85; 2444-2448 (1988). The BLAST family of programs is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well known Smith Waterman algorithm may also be usedto determine identity.

[0180] Parameters for polypeptide sequence comparison include thefollowing:

[0181] Algorithm: Needleman and Wunsch, J. Mol Biol. 48: 443-453 (1970)

[0182] Comparison matrix: BLOSSUM62 from Henikoff and Henikoff,

[0183] Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992)

[0184] Gap Penalty: 8

[0185] Gap Length Penalty: 2

[0186] A program useful with these parameters is publicly available asthe “gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidecomparisons (along with no penalty for end gaps).

[0187] Parameters for polynucleotide comparison include the following:

[0188] Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)

[0189] Comparison matrix: matches=+10, mismatch=0

[0190] Gap Penalty: 50

[0191] Gap Length Penalty: 3

[0192] Available as: The “gap” program from Genetics Computer Group,Madison Wis.

[0193] These are the default parameters for nucleic acid comparisons.

[0194] A preferred meaning for “identity” for polynucleotides andpolypeptides, as the case may be, are provided in (1) and (2) below.

[0195] (1) Polynucleotide embodiments further include an isolatedpolynucleotide comprising a polynucleotide sequence having at least a50, 60, 70, 80, 85, 90, 95, 97 or 100% identity to the referencesequence of SEQ ID NO: 1, wherein said polynucleotide sequence may beidentical to the reference sequence of SEQ ID NO: 1 or may include up toa certain integer number of nucleotide alterations as compared to thereference sequence, wherein said alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein said alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence, andwherein said number of nucleotide alterations is determined bymultiplying the total number of nucleotides in SEQ ID NO: 1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleotides in SEQ IDNO:1, or:

n _(n) ≦x _(n)−(x _(n) ∘ y)

[0196] wherein n_(n) is the number of nucleotide alterations, x_(n) isthe total number of nucleotides in SEQ ID NO: 1, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and ∘ is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n). Alterations of a polynucleotide sequence encoding thepolypeptide of SEQ ID NO:2 may create nonsense, missense or frameshiftmutations in this coding sequence and thereby alter the polypeptideencoded by the polynucleotide following such alterations.

[0197] By way of example, a polynucleotide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO: 1,that is it may be 100% identical, or it may include up to a certaininteger number of nucleic acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least onenucleic acid deletion, substitution, including transition andtransversion, or insertion, and wherein said alterations may occur atthe 5′ or 3′ terminal positions of the reference polynucleotide sequenceor anywhere between those terminal positions, interspersed eitherindividually among the nucleic acids in the reference sequence or in oneor more contiguous groups within the reference sequence. The number ofnucleic acid alterations for a given percent identity is determined bymultiplying the total number of nucleic acids in SEQ ID NO: 1 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of nucleic acids in SEQID NO: 1, or:

n _(n) ≦x _(n)−(x _(n) ∘ y)

[0198] wherein n_(n) is the number of nucleic acid alterations, x_(n) isthe total number of nucleic acids in SEQ ID NO: 1, y is, for instance0.70 for 70%, 0.80 for 80%, 0.85 for 85% etc., ∘ is the symbol for themultiplication operator, and wherein any non-integer product of x_(n)and y is rounded down to the nearest integer prior to subtracting itfrom x_(n).

[0199] (2) Polypeptide embodiments further include an isolatedpolypeptide comprising a polypeptide having at least a 50, 60, 70, 80,85, 90, 95, 97 or 100% identity to a polypeptide reference sequence ofSEQ ID NO:2, wherein said polypeptide sequence may be identical to thereference sequence of SEQ ID NO:2 or may include up to a certain integernumber of amino acid alterations as compared to the reference sequence,wherein said alterations are selected from the group consisting of atleast one amino acid deletion, substitution, including conservative andnon-conservative substitution, or insertion, and wherein saidalterations may occur at the amino- or carboxy-terminal positions of thereference polypeptide sequence or anywhere between those terminalpositions, interspersed either individually among the amino acids in thereference sequence or in one or more contiguous groups within thereference sequence, and wherein said number of amino acid alterations isdetermined by multiplying the total number of amino acids in SEQ ID NO:2by the integer defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ∘ y)

[0200] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is 0.50 for 50%, 0.60for 60%, 0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95for 95%, 0.97 for 97% or 1.00 for 100%, and ∘ is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0201] By way of example, a polypeptide sequence of the presentinvention may be identical to the reference sequence of SEQ ID NO:2,that is it may be 100% identical, or it may include up to a certaininteger number of amino acid alterations as compared to the referencesequence such that the percent identity is less than 100% identity. Suchalterations are selected from the group consisting of at least one aminoacid deletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein said alterations may occur atthe amino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO:2 by theinteger defining the percent identity divided by 100 and thensubtracting that product from said total number of amino acids in SEQ IDNO:2, or:

n _(a) ≦x _(a)−(x _(a) ∘ y)

[0202] wherein n_(a) is the number of amino acid alterations, x_(a) isthe total number of amino acids in SEQ ID NO:2, y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and ∘ is the symbol for themultiplication operator, and wherein any non-integer product of x_(a)and y is rounded down to the nearest integer prior to subtracting itfrom x_(a).

[0203] “Individual(s),” when used herein with reference to an organism,means a multicellular eukaryote, including, but not limited to ametazoan, a mammal, an ovid, a bovid, a simian, a primate, and a human.

[0204] “Isolated” means altered “by the hand of man” from its naturalstate, i.e., if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. Moreover, a polynucleotide or polypeptide that is introducedinto an organism by transformation, genetic manipulation or by any otherrecombinant method is “isolated” even if it is still present in saidorganism, which organism may be living or non-living.

[0205] “Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA including single and double-stranded regions.

[0206] “Variant” refers to a polynucleotide or polypeptide that differsfrom a reference polynucleotide or polypeptide, but retains essentialproperties. A typical variant of a polynucleotide differs in nucleotidesequence from another, reference polynucleotide. Changes in thenucleotide sequence of the variant may or may not alter the amino acidsequence of a polypeptide encoded by the reference polynucleotide.Nucleotide changes may result in amino acid substitutions, additions,deletions, fusions and truncations in the polypeptide encoded by thereference sequence, as discussed below. A typical variant of apolypeptide differs in amino acid sequence from another, referencepolypeptide. Generally, differences are limited so that the sequences ofthe reference polypeptide and the variant are closely similar overalland, in many regions, identical. A variant and reference polypeptide maydiffer in amino acid sequence by one or more substitutions, additions,deletions in any combination. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code. A variant ofa polynucleotide or polypeptide may be a naturally occurring such as anallelic variant, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

[0207] “Disease(s)” means any disease caused by or related to infectionby a bacteria, including, for example, upper respiratory tractinfection, invasive bacterial diseases, such as bacteremia andmeningitis.

EXAMPLES

[0208] The examples below are carried out using standard techniques,which are well known and routine to those of skill in the art, exceptwhere otherwise described in detail. The examples are illustrative, butdo not limit the invention.

Example 1 Discovery and Confirmatory DNA Sequencing of the BASB013 Genefrom two N. menineitidis Isolates.

[0209] A: BASB013 in N. meningitidis Serogroup B Strain ATCC13090.

[0210] The BASB013 gene disclosed in SEQ ID NO:1 was first discovered inthe Incyte PathoSeq database containing unfinished genomic DNA sequencesof the N. meningitidis strain ATCC 13090. The translation of the BASB013polynucleotide sequence, shown in SEQ ID NO:2, showed significantsimilarity (51% identity in a 456 amino acids overlap) to the MucDprotein of Pseudomonas aeruginosa, the latter being a homolog of theHtrA serine protease found in many bacteria, in particular inEscherichia coli and Haemophilus influenzae.

[0211] The sequence of the BASB013 gene was further confirmedexperimentally. For this purpose, genomic DNA was extracted from 10¹⁰cells of the N. meningitidis cells (strain ATCC 13090) using the QIAGENgenomic DNA extraction kit (Qiagen Gmbh), and 1 μg of this material wassubmitted to Polymerase Chain Reaction DNA amplification using primersHin-full-N (5′-GGA ATT CCA TAT GTT CAA AAA ATA CCA ATA CC-3′) [SEQ IDNO:11] and Hin-full-X (5′-CGC CGC TCG AGT TGC AGG TTT AAT GCG ATG-3′)[SEQ ID NO:12]. This PCR product was gel-purified and subjected to DNAsequencing using the Big Dye Cycle Sequencing kit (Perkin-Elmer) and anABI 373A/PRISM DNA sequencer. DNA sequencing was performed on bothstrands with a redundancy of 2 and the full-length sequence wasassembled using the Seqman program from the DNASTAR Lasergene softwarepackage. The resulting DNA sequence and deduced peptide sequence areshown as SEQ ID NO:3 and SEQ ID NO:4 respectively. Three nucleotides inSEQ ID NO:3 (at positions 615 to 617) were found different from theircounterpart in SEQ ID NO:1, as shown in FIG. 1.

[0212] It should be noted that the start codon of the BASB013 gene isGTG, which is not unusual in bacterial genes. That GTG codon has beentranslated as Methionine.

[0213] B: BASB013 in N. meningitidis Serogroup B Strain H44/76.

[0214] The sequence of the BASB013 gene was also determined in an otherN. meningitidis serogroup B strain, the strain H44/76. For this purpose,genomic DNA was extracted from the N. meningitidis strain H44/76 usingthe experimental conditions presented in Example 1. This material (1 μg)was then submitted to Polymerase Chain Reaction DNA amplification usingprimers Hin-full-N and Hin-full-X specific for the BASB013 gene. A 1518bp DNA fragment was obtained, digested by the NdeI/XhoI restrictionendonucleases and inserted into the corresponding sites of the pET-24bcloning/expression vector (Novagen) using standard molecular biologytechniques (Molecular Cloning, a Laboratory Manual, Second Edition, Eds:Sambrook, Fritsch & Maniatis, Cold Spring Harbor press 1989).Recombinant pET-24b/BASB013 was then submitted to DNA sequencing usingthe Big Dyes kit (Applied biosystems) and analyzed on a ABI 373/A DNAsequencer in the conditions described by the supplier. As a result, thepolynucleotide and deduced polypeptide sequences, referred to as SEQ IDNO:5 and SEQ ID NO:6 respectively, were obtained.

[0215] Using the PILEUP program from the GCG package, a multiplealignment of the nucleotide sequences of SEQ ID NO: 1, 3 and 5 wasperformed, and is displayed in FIG. 1. A pairwise comparison ofidentities is summarized in Table 1, showing that the three BASB013polynucleotide gene sequences are all similar at a identity levelgreater than or equal to 96.2%. Using the same PILEUP program from theGCG package, a multiple alignment of the protein sequences of SEQ IDNO:2, 4 and 6 was performed, and is displayed in FIG. 2. A pairwisecomparison of identities is summarized in Table 2, showing that thethree BASB013 polypeptide sequences are all similar at a identity levelequal to or greater than 95.8%.

[0216] Taken together, these data indicate strong sequence conservationof the BASB013 gene among the two N. meningitidis serogroup B strains.

Example 2 Expression and Purification of Recombinant BASB013 Protein inEscherichia coli.

[0217] As represented in FIG. 1, nucleotide sequence comparison betweentwo N. menigitidis strains indicated that the first 1110 nucleotides ofBASB013 shared a high degree of sequence conservation. In contrast, thelast 390 nucleotides of the BASB013 gene showed some sequencevariability. Consequently, expression vectors allowing the production ofthe full-length, the conserved (BASB013-C, [SEQ ID NO:7] and [SEQ IDNO:8]) or the variable (BASB013-V, [SEQ ID NO:9] and [SEQ ID NO: 10])part were constructed. The construction of the pET-24bcloning/expression vector containing the full-length BASB013 gene wasdescribed in Example 1B. The BASB013-C fragment was amplified by PCRusing oligonucleotide primers HC1 (5′-GAT ATA CAT ATG TTC AAA AAA TACCAA TAC CTC-3′) [SEQ ID NO: 13] and HC2 (5′-CTA GGG CTC GAG TCC CGG CGTAAT GGC GCC GAC-3′) [SEQ ID NO: 14]. The BASB013-V fragment wasamplified by PCR using oligonucleotide primers HV1 (5′-GAT ATA CAT ATGAAA GAA GTC AGC CTC GGC GTA-3′) [SEQ ID NO:15] and HV2 (5′-CTA GGG CTCGAG TTG CAG GTT TAA TGC GAT GAA-3′) [SEQ ID NO:16]. Both PCR ampliconswere digested using NdeI and XhoI and inserted in the correspondingsites of the pET24b cloning/expression vector using standard molecularbiology methodology.

[0218] For these constructs, the BASB013, the BASB013-C and theBASB013-V genes were isolated from the strain H44/76. These genes areintroduced in fusion with a stretch of 6 histidine residues, and areplaced under the control of the strong bacteriophage T7 gene 10promoter. For expression study, this vector was introduced into theEscherichia coli strain BL21 DE3 (Novagen), in which, the gene for theT7 polymerase is placed under the control of the isopropyl-beta-Dthiogalactoside (IPTG)-regulatable lac promoter. Liquid cultures (100ml) of the BL21 DE-3 [pET-24b/BASB013], BL21 DE-3 [pET-24b/BASB013-C]and BL21 DE-3 [pET-24b/BASB013-V] E. coli recombinant strains were grownat 37° C. under agitation until the optical density at 600 nm (OD600)reached 0.6. At that time-point, IPTG was added at a final concentrationof 1 mM and the culture was grown for 4 additional hours. The culturewas then centrifuged at 10.000 rpm and the pellet was frozen at −20° C.for at least 10 hours. Subcellular localization of the polypeptideexpressed from pET24b indicated that the BASB013 remained soluble in thebacterial cytoplasm whereas BASB013-C and BASB013-V were insoluble.Consequently, BASB013 was purified under mild, non-denaturing conditionswhereas BASB013-C and BASB013-V were purified under denaturingconditions.

[0219] Purification of BASB013 Under Mild, Non-Denaturing Conditions:

[0220] After thawing, the cell paste was resuspended in 12.5 ml of startbuffer (10 mM sodium-phosphate pH7.4, NaCl 0.5M, Imidazole 10 mM)containing 100 mM of Pefablock (Boehringer Mannheim) protease inhibitor.The sample was then loaded at a flow-rate of 1 ml/min on a Ni2+-loadedHitrap column (Pharmacia Biotech). After passsage of the flow trough,the column was washed succesively with start buffer (40 ml) and startbuffer containing 60 mM Imidazole (30 ml). The recombinant proteinBASB013/His6 was then eluted from the column with 30 ml of start buffercontaining 500 mM of imidazole and 3 ml-size fractions were collected.

[0221] Purification of BASB013-C and BASB013-V Under DenaturingConditions:

[0222] After thawing, the cell paste was resuspended during 30 min at25° C. in buffer A (6M guanidine hydrochloride, 0.1M NaH₂PO₄, 0.01MTris, pH 8.0), passed three-times through a needle and clarified bycentrifugation (20000 rpm, 15 min). The sample was then loaded at aflow-rate of 1 ml/min on a Ni2+-loaded Hitrap column (PharmaciaBiotech). After passsage of the flowthrough, the column was washedsuccesively with 40 ml of buffer B (8M Urea, 0.1 M NaH₂PO₄, 0.01 M Tris,pH 8.0), 40 ml of buffer C (8M Urea, 0.1M NaH₂PO₄, 0.01 M Tris, pH 6.3).The recombinant protein BASB013/His6 was then eluted from the columnwith 30 ml of buffer C (8M Urea, 0.1M NaH₂PO₄, 0.01M Tris, pH 6.3)containing 500 mM of imidazole and 3 ml-size fractions were collected.As shown in FIG. 3, enriched fractions (purity estimated to more than80% pure in coomassie staining) were obtained for BASB013 (MW estimatedto 52 kDa), and BASB013-V (MW estimated to 14 kDa) after elution fromthe column. In FIG. 3, substantially purified proteins were separated ona 4-20% gradient polyacrylamide gel (NOVEX) under SDS-PAGE conditionsand stained with Coomassie Blue R250. The sample loaded on the gelcorresponded to molecular weight marker (lanes 1 and 4) and proteinfractions enriched (more than 80%) in BASB013 (lane 2) and BASB013-V(lane3). For BASB013-C (MW estimated to 40 kDa), the recovery yield wasvery low and the protein was not detected by coomassie staining andrequired western blotting analysis (data not shown). These 3polypeptides were reactive against a mouse monoclonal antibody raisedagainst the 5-histidine motif. Taken together, these data indicate thatthe BASB013 gene can be expressed and purified under several recombinantforms (BASB013/His6, BASB013—C/His6 and BASB013-V/His6) in E. coli.TABLE 1 Pairwise identities off the BASB013 polynucleotide sequences (in%) SeqID No: 3 SeqID No: 5 SeqID No: 1 99.8 96.2 SeqID No: 3 96.4

[0223] TABLE 2 Pairwise identities off the BASB013 polypeptide sequences(in %) SeqID No: 4 SeqID No: 6 SeqID No: 2 99.8 95.8 SeqID No: 4 96.0

Example 3 Presence of Anti-BASB013 Antibodies in Sera From ConvalescentPatients.

[0224] In this test, human convalescent sera have been tested bywestern-blotting for recognition of the purified recombinant BASB013protein.

[0225] 15 μg of purified BASB013 protein (full length, cl. 8) are putinto a SDS-PAGE gradient gel (4-20%, Novex, code n^(o)EC60252) forelectrophoretic migration. Proteins are transferred to nitrocellulosesheet (0.45 μm, Bio-rad code n^(o) 162-0114) at 100 volts for 1 hourusing a Bio-rad Trans-blot system (code n^(o) 170-3930). The filter isthen blocked with PBS-0.05% Tween 20 overnight at room temperature,before incubation with the human sera. These sera are diluted 100 foldin PBS-0.05% Tween 20, and incubated on the nitrocellulose sheet for twohours at room temperature with gentle shaking, using a mini-blottersystem (Miniprotean, Bio-rad code n^(o) 170-4017). After three repeatedwashing steps in PBS-0.05% Tween 20 for 5 min., the nitrocellulose sheetis incubated at room temperature for 1 hour under gentle shaking withthe appropriate conjugate (biotinylated anti-human Ig antibodies fromsheep, Amersham code n^(o)RPN1003) diluted at 1/500 in the same washingbuffer. The membrane is washed three times as previously, and incubatedfor 30 min. with agitation using the streptavidin-peroxidase complex(Amersham code n^(o)1051) diluted at 1/1000 in the washing buffer. Afterthe last three repeated washing steps, the revelation occurs during the20 min incubation time in a 50 ml solution containing 30 mg4-chloro-1-naphtol (Sigma), 10 ml methanol, 40 ml of ultra-pure water,and 30 μl of H₂O₂. The staining is stopped while washing the membraneseveral times in distillated water. In part A of the western-blot,revelation was done with a pool of mice sera from animals immunized with2 μg of Outer Membrane Proteins (OMP, obtained by Lithium Chlorideextraction) injected in SB62 with 5 μg MPL and 2 μg QS21 by thesubcutaneous and intraperitoneal routes on days 0, 21 and 42. Animalswere bled on day 49. Mice antibodies were detected as for humanantibodies except the conjugate used was a biotinylated anti-mouse Igantibodies from sheep, Amersham code n^(o)RPN100I) diluted at 1/500.

[0226] Results illustrated in FIG. 4 (Part B) show that 6/7 convalescentsera tested react against the purified recombinant BASB013 protein (seearrow; only patient n^(o) 261324 does not show any reactivity againstthe protein). The BASB013 band is clearly visible at around 53 kD. Inpart A of the western-blot, it is seen that mice immunized with OMP fromstrain H44/76 recognize very well the recombinant BASB013 polypeptide atthe same molecular weight (53 kD).

[0227] Deposited Materials

[0228] A deposit containing a Neisseria meningitidis Serogroup B strainhas been deposited with the American Type Culture Collection (herein“ATCC”) on Jun. 22, 1997 and assigned deposit number 13090. The depositwas described as Neisseria Meningitidis (Albrecht and Ghon) and is afreeze-dried, 1.5-2.9 kb insert library constructed from N. memingitidisisolate. The deposit is described in Int. Bull. Bacteriol. Nomencl.Taxon. 8: 1-15 (1958).

[0229] The Neisseria meningitidis strain deposit is referred to hereinas “the deposited strain” or as “the DNA of the deposited strain.”

[0230] The deposited strain contains the full length BASB013 gene. Thesequence of the polynucleotides contained in the deposited strain, aswell as the amino acid sequence of any polypeptide encoded thereby, arecontrolling in the event of any conflict with any description ofsequences herein.

[0231] The deposit of the deposited strain has been made under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for Purposes of Patent Procedure. The strain will beirrevocably and without restriction or condition released to the publicupon the issuance of a patent. The deposited strain is provided merelyas convenience to those of skill in the art and is not an admission thata deposit is required for enablement, such as that required under 35U.S.C. §112.

1 16 1 1500 DNA Bacteria 1 gtgttcaaaa aataccaata cctcgctttg gcagcactgtgtgccgcctc gctggcaggc 60 tgcgacaaag ccggcagctt tttcggtgcg gacaaaaaagaagcatcctt cgtagaacgc 120 atcgaacaca ccaaagacga cggcagcgtc agtatgctgctgcccgactt tgtccaactg 180 gttcaaagcg aaggcccggc agtcgtcaat attcaggcagcccccgcccc gcgcacccaa 240 aacggcagcg gcaatgccga aaccgattcc gacccgcttgccgacagcga cccgttctac 300 gaatttttca aacgcctcgt cccgaatatg cccgaaatcccccaagaaga agcagatgac 360 ggcggattga acttcggttc gggcttcatc atcagcaaagacggctatat tctgaccaat 420 acgcacgtcg ttaccggcat gggcagtatc aaagtcctgctcaacgacaa gcgcgaatat 480 accgccaaac tcatcggttc ggatgtccaa tccgatgtcgcccttctgaa aatcgacgca 540 acggaagagc tgcccgtcgt caaaatcggc aatcccaaagatttgaaacc gggcgaatgg 600 gtcgccgcca tcgggcggcc cttcggcttc gacaacagcgtgaccgccgg catcgtgtcc 660 gccaaaggca gaagcctgcc caacgaaagc tacacacccttcatccaaac cgacgttgcc 720 atcaatccgg gcaactccgg cggcccgctg ttcaacctgaaaggacaggt cgtcggcatc 780 aactcgcaaa tatacagccg cagcggcgga ttcatgggcatttccttcgc catcccgatt 840 gacgttgcca tgaatgtcgc cgaacagctg aaaaacaccggcaaagtcca acgcggacaa 900 ctgggcgtga ttattcaaga agtatcctac ggtttggcacaatcgttcgg tttggacaaa 960 gccggcggcg cactgattgc caaaatcctg cccggcagccccgcagaacg tgccggcctg 1020 caggcgggcg acatcgtcct cagcctcgac ggcggagaaatacgttcttc cggcgacctt 1080 cccgttatgg tcggcgccat tacgccggga aaagaagtcagcctcggcgt atggcgcaaa 1140 ggtaaggaaa tcaccgttgc cgtcaaactg ggcaatgcttccgaacaaac cggttcctcg 1200 tccgagccgg acaaagcccc ttatgccgaa caccaatccggtacgttctc ggtcgaatcc 1260 gcaggcatta cccttcagac acataccgac agcagcggcggacggcttgt cgtcgtgcgg 1320 gtttcggggg cggcagaacg cgcaggcttg aggcgcggcgacgaaatcct tgccgtcggg 1380 caagtccccg tcaatgacga agacggtttc cgcaaagctatggacaaggc aggcaaaaac 1440 gtccccctgc tggtcatgcg ccgtggcaac acgctgttcatcgcattaaa cctgcaataa 1500 2 499 PRT Bacteria 2 Met Phe Lys Lys Tyr GlnTyr Leu Ala Leu Ala Ala Leu Cys Ala Ala 1 5 10 15 Ser Leu Ala Gly CysAsp Lys Ala Gly Ser Phe Phe Gly Ala Asp Lys 20 25 30 Lys Glu Ala Ser PheVal Glu Arg Ile Glu His Thr Lys Asp Asp Gly 35 40 45 Ser Val Ser Met LeuLeu Pro Asp Phe Val Gln Leu Val Gln Ser Glu 50 55 60 Gly Pro Ala Val ValAsn Ile Gln Ala Ala Pro Ala Pro Arg Thr Gln 65 70 75 80 Asn Gly Ser GlyAsn Ala Glu Thr Asp Ser Asp Pro Leu Ala Asp Ser 85 90 95 Asp Pro Phe TyrGlu Phe Phe Lys Arg Leu Val Pro Asn Met Pro Glu 100 105 110 Ile Pro GlnGlu Glu Ala Asp Asp Gly Gly Leu Asn Phe Gly Ser Gly 115 120 125 Phe IleIle Ser Lys Asp Gly Tyr Ile Leu Thr Asn Thr His Val Val 130 135 140 ThrGly Met Gly Ser Ile Lys Val Leu Leu Asn Asp Lys Arg Glu Tyr 145 150 155160 Thr Ala Lys Leu Ile Gly Ser Asp Val Gln Ser Asp Val Ala Leu Leu 165170 175 Lys Ile Asp Ala Thr Glu Glu Leu Pro Val Val Lys Ile Gly Asn Pro180 185 190 Lys Asp Leu Lys Pro Gly Glu Trp Val Ala Ala Ile Gly Arg ProPhe 195 200 205 Gly Phe Asp Asn Ser Val Thr Ala Gly Ile Val Ser Ala LysGly Arg 210 215 220 Ser Leu Pro Asn Glu Ser Tyr Thr Pro Phe Ile Gln ThrAsp Val Ala 225 230 235 240 Ile Asn Pro Gly Asn Ser Gly Gly Pro Leu PheAsn Leu Lys Gly Gln 245 250 255 Val Val Gly Ile Asn Ser Gln Ile Tyr SerArg Ser Gly Gly Phe Met 260 265 270 Gly Ile Ser Phe Ala Ile Pro Ile AspVal Ala Met Asn Val Ala Glu 275 280 285 Gln Leu Lys Asn Thr Gly Lys ValGln Arg Gly Gln Leu Gly Val Ile 290 295 300 Ile Gln Glu Val Ser Tyr GlyLeu Ala Gln Ser Phe Gly Leu Asp Lys 305 310 315 320 Ala Gly Gly Ala LeuIle Ala Lys Ile Leu Pro Gly Ser Pro Ala Glu 325 330 335 Arg Ala Gly LeuGln Ala Gly Asp Ile Val Leu Ser Leu Asp Gly Gly 340 345 350 Glu Ile ArgSer Ser Gly Asp Leu Pro Val Met Val Gly Ala Ile Thr 355 360 365 Pro GlyLys Glu Val Ser Leu Gly Val Trp Arg Lys Gly Lys Glu Ile 370 375 380 ThrVal Ala Val Lys Leu Gly Asn Ala Ser Glu Gln Thr Gly Ser Ser 385 390 395400 Ser Glu Pro Asp Lys Ala Pro Tyr Ala Glu His Gln Ser Gly Thr Phe 405410 415 Ser Val Glu Ser Ala Gly Ile Thr Leu Gln Thr His Thr Asp Ser Ser420 425 430 Gly Gly Arg Leu Val Val Val Arg Val Ser Gly Ala Ala Glu ArgAla 435 440 445 Gly Leu Arg Arg Gly Asp Glu Ile Leu Ala Val Gly Gln ValPro Val 450 455 460 Asn Asp Glu Asp Gly Phe Arg Lys Ala Met Asp Lys AlaGly Lys Asn 465 470 475 480 Val Pro Leu Leu Val Met Arg Arg Gly Asn ThrLeu Phe Ile Ala Leu 485 490 495 Asn Leu Gln 3 1500 DNA Bacteria 3gtgttcaaaa aataccaata cctcgctttg gcagcactgt gtgccgcctc gctggcaggc 60tgcgacaaag ccggcagctt tttcggtgcg gacaaaaaag aagcatcctt cgtagaacgc 120atcgaacaca ccaaagacga cggcagcgtc agtatgctgc tgcccgactt tgtccaactg 180gttcaaagcg aaggcccggc agtcgtcaat attcaggcag cccccgcccc gcgcacccaa 240aacggcagcg gcaatgccga aaccgattcc gacccgcttg ccgacagcga cccgttctac 300gaatttttca aacgcctcgt cccgaatatg cccgaaatcc cccaagaaga agcagatgac 360ggcggattga acttcggttc gggcttcatc atcagcaaag acggctatat tctgaccaat 420acgcacgtcg ttaccggcat gggcagtatc aaagtcctgc tcaacgacaa gcgcgaatat 480accgccaaac tcatcggttc ggatgtccaa tccgatgtcg cccttctgaa aatcgacgca 540acggaagagc tgcccgtcgt caaaatcggc aatcccaaag atttgaaacc gggcgaatgg 600gtcgccgcca tcggcgcgcc cttcggcttc gacaacagcg tgaccgccgg catcgtgtcc 660gccaaaggca gaagcctgcc caacgaaagc tacacaccct tcatccaaac cgacgttgcc 720atcaatccgg gcaactccgg cggcccgctg ttcaacctga aaggacaggt cgtcggcatc 780aactcgcaaa tatacagccg cagcggcgga ttcatgggca tttccttcgc catcccgatt 840gacgttgcca tgaatgtcgc cgaacagctg aaaaacaccg gcaaagtcca acgcggacaa 900ctgggcgtga ttattcaaga agtatcctac ggtttggcac aatcgttcgg tttggacaaa 960gccggcggcg cactgattgc caaaatcctg cccggcagcc ccgcagaacg tgccggcctg 1020caggcgggcg acatcgtcct cagcctcgac ggcggagaaa tacgttcttc cggcgacctt 1080cccgttatgg tcggcgccat tacgccggga aaagaagtca gcctcggcgt atggcgcaaa 1140ggtaaggaaa tcaccgttgc cgtcaaactg ggcaatgctt ccgaacaaac cggttcctcg 1200tccgagccgg acaaagcccc ttatgccgaa caccaatccg gtacgttctc ggtcgaatcc 1260gcaggcatta cccttcagac acataccgac agcagcggcg gacggcttgt cgtcgtgcgg 1320gtttcggggg cggcagaacg cgcaggcttg aggcgcggcg acgaaatcct tgccgtcggg 1380caagtccccg tcaatgacga agacggtttc cgcaaagcta tggacaaggc aggcaaaaac 1440gtccccctgc tggtcatgcg ccgtggcaac acgctgttca tcgcattaaa cctgcaataa 1500 4499 PRT Bacteria 4 Met Phe Lys Lys Tyr Gln Tyr Leu Ala Leu Ala Ala LeuCys Ala Ala 1 5 10 15 Ser Leu Ala Gly Cys Asp Lys Ala Gly Ser Phe PheGly Ala Asp Lys 20 25 30 Lys Glu Ala Ser Phe Val Glu Arg Ile Glu His ThrLys Asp Asp Gly 35 40 45 Ser Val Ser Met Leu Leu Pro Asp Phe Val Gln LeuVal Gln Ser Glu 50 55 60 Gly Pro Ala Val Val Asn Ile Gln Ala Ala Pro AlaPro Arg Thr Gln 65 70 75 80 Asn Gly Ser Gly Asn Ala Glu Thr Asp Ser AspPro Leu Ala Asp Ser 85 90 95 Asp Pro Phe Tyr Glu Phe Phe Lys Arg Leu ValPro Asn Met Pro Glu 100 105 110 Ile Pro Gln Glu Glu Ala Asp Asp Gly GlyLeu Asn Phe Gly Ser Gly 115 120 125 Phe Ile Ile Ser Lys Asp Gly Tyr IleLeu Thr Asn Thr His Val Val 130 135 140 Thr Gly Met Gly Ser Ile Lys ValLeu Leu Asn Asp Lys Arg Glu Tyr 145 150 155 160 Thr Ala Lys Leu Ile GlySer Asp Val Gln Ser Asp Val Ala Leu Leu 165 170 175 Lys Ile Asp Ala ThrGlu Glu Leu Pro Val Val Lys Ile Gly Asn Pro 180 185 190 Lys Asp Leu LysPro Gly Glu Trp Val Ala Ala Ile Gly Ala Pro Phe 195 200 205 Gly Phe AspAsn Ser Val Thr Ala Gly Ile Val Ser Ala Lys Gly Arg 210 215 220 Ser LeuPro Asn Glu Ser Tyr Thr Pro Phe Ile Gln Thr Asp Val Ala 225 230 235 240Ile Asn Pro Gly Asn Ser Gly Gly Pro Leu Phe Asn Leu Lys Gly Gln 245 250255 Val Val Gly Ile Asn Ser Gln Ile Tyr Ser Arg Ser Gly Gly Phe Met 260265 270 Gly Ile Ser Phe Ala Ile Pro Ile Asp Val Ala Met Asn Val Ala Glu275 280 285 Gln Leu Lys Asn Thr Gly Lys Val Gln Arg Gly Gln Leu Gly ValIle 290 295 300 Ile Gln Glu Val Ser Tyr Gly Leu Ala Gln Ser Phe Gly LeuAsp Lys 305 310 315 320 Ala Gly Gly Ala Leu Ile Ala Lys Ile Leu Pro GlySer Pro Ala Glu 325 330 335 Arg Ala Gly Leu Gln Ala Gly Asp Ile Val LeuSer Leu Asp Gly Gly 340 345 350 Glu Ile Arg Ser Ser Gly Asp Leu Pro ValMet Val Gly Ala Ile Thr 355 360 365 Pro Gly Lys Glu Val Ser Leu Gly ValTrp Arg Lys Gly Lys Glu Ile 370 375 380 Thr Val Ala Val Lys Leu Gly AsnAla Ser Glu Gln Thr Gly Ser Ser 385 390 395 400 Ser Glu Pro Asp Lys AlaPro Tyr Ala Glu His Gln Ser Gly Thr Phe 405 410 415 Ser Val Glu Ser AlaGly Ile Thr Leu Gln Thr His Thr Asp Ser Ser 420 425 430 Gly Gly Arg LeuVal Val Val Arg Val Ser Gly Ala Ala Glu Arg Ala 435 440 445 Gly Leu ArgArg Gly Asp Glu Ile Leu Ala Val Gly Gln Val Pro Val 450 455 460 Asn AspGlu Asp Gly Phe Arg Lys Ala Met Asp Lys Ala Gly Lys Asn 465 470 475 480Val Pro Leu Leu Val Met Arg Arg Gly Asn Thr Leu Phe Ile Ala Leu 485 490495 Asn Leu Gln 5 1500 DNA Bacteria 5 gtgttcaaaa aataccaata cctcgctttggcagcactgt gtgccgcctc gctggcaggc 60 tgcgacaagg caggcagctt tttcggtgcggacaaaaaag aagcatcctt cgtagaacgc 120 atcgaacaca ccaaagacga cggcagcgtcagtatgctgc tgcccgactt tgcccaactg 180 gttcaaagtg aaggtccggc agtcgtcaatattcaggcag cccccgcccc gcgcacccaa 240 aacggcagcg gcaatgccga aaacgattccgacccgattg ccgacaacga cccgttctac 300 gaatttttca aacgcctcgt cccgaatatgcccgaaatcc cccaagaaga agcagatgac 360 ggcggattga acttcggttc gggcttcatcatcagcaaag acggctacat cctgaccaat 420 acccacgtcg ttaccggcat gggcagtatcaaagtcctgc tcaacgacaa gcgcgaatat 480 accgccaaac tcatcggttc ggatgtccaatccgatgtcg cccttctgaa aatcgacgca 540 acggaagagc tgcccgtcgt caaaatcggcaatcccaaag atttgaaacc gggcgaatgg 600 gtcgccgcca tcggcgcgcc cttcggcttcgacaacagcg tgaccgccgg catcgtgtcc 660 gccaaaggca gaagcctgcc caacgaaagctacacaccct tcatccaaac cgacgttgcc 720 atcaatccgg gcaactccgg cggcccgctgttcaacttaa aaggacaggt cgtcggcatc 780 aactcgcaaa tatacagccg cagcggcggattcatgggca tttccttcgc catcccgatt 840 gacgttgcca tgaatgtcgc cgaacagctgaaaaacaccg gcaaagtcca acgcggacaa 900 ctgggcgtga ttattcaaga agtatcctacggtttggcac aatcgttcgg cttggacaaa 960 gccggcggcg cactgattgc caaaatcctgcccggcagcc ccgcagaacg tgccggcctg 1020 caggcgggcg acatcgtcct cagcctcgacggcggagaaa tacgttcttc cggcgacctt 1080 cccgttatgg tcggcgccat tacgccgggaaaagaagtca gcctcggcgt atggcgcaaa 1140 ggcgaagaaa tcacaatcaa agtcaagctgggcaacgccg ccgagcatat cggcgcatca 1200 tccaaaacag atgaagcccc ctacaccgaacagcaatccg gtacgttctc ggtcgaatcc 1260 gcaggcatta cccttcagac acataccgacagcagcggcg gacacctcgt cgtcgtacgg 1320 gtttccgacg cggcagaacg cgcaggcttgaggcgcggcg acgaaattct tgccgtcggg 1380 caagtccccg tcaatgacga agccggtttccgcaaagcta tggacaaggc aggcaaaaac 1440 gtccccctgc tgatcatgcg ccgtggcaacacgctgttca tcgcattaaa cctgcaataa 1500 6 499 PRT Bacteria 6 Met Phe LysLys Tyr Gln Tyr Leu Ala Leu Ala Ala Leu Cys Ala Ala 1 5 10 15 Ser LeuAla Gly Cys Asp Lys Ala Gly Ser Phe Phe Gly Ala Asp Lys 20 25 30 Lys GluAla Ser Phe Val Glu Arg Ile Glu His Thr Lys Asp Asp Gly 35 40 45 Ser ValSer Met Leu Leu Pro Asp Phe Ala Gln Leu Val Gln Ser Glu 50 55 60 Gly ProAla Val Val Asn Ile Gln Ala Ala Pro Ala Pro Arg Thr Gln 65 70 75 80 AsnGly Ser Gly Asn Ala Glu Asn Asp Ser Asp Pro Ile Ala Asp Asn 85 90 95 AspPro Phe Tyr Glu Phe Phe Lys Arg Leu Val Pro Asn Met Pro Glu 100 105 110Ile Pro Gln Glu Glu Ala Asp Asp Gly Gly Leu Asn Phe Gly Ser Gly 115 120125 Phe Ile Ile Ser Lys Asp Gly Tyr Ile Leu Thr Asn Thr His Val Val 130135 140 Thr Gly Met Gly Ser Ile Lys Val Leu Leu Asn Asp Lys Arg Glu Tyr145 150 155 160 Thr Ala Lys Leu Ile Gly Ser Asp Val Gln Ser Asp Val AlaLeu Leu 165 170 175 Lys Ile Asp Ala Thr Glu Glu Leu Pro Val Val Lys IleGly Asn Pro 180 185 190 Lys Asp Leu Lys Pro Gly Glu Trp Val Ala Ala IleGly Ala Pro Phe 195 200 205 Gly Phe Asp Asn Ser Val Thr Ala Gly Ile ValSer Ala Lys Gly Arg 210 215 220 Ser Leu Pro Asn Glu Ser Tyr Thr Pro PheIle Gln Thr Asp Val Ala 225 230 235 240 Ile Asn Pro Gly Asn Ser Gly GlyPro Leu Phe Asn Leu Lys Gly Gln 245 250 255 Val Val Gly Ile Asn Ser GlnIle Tyr Ser Arg Ser Gly Gly Phe Met 260 265 270 Gly Ile Ser Phe Ala IlePro Ile Asp Val Ala Met Asn Val Ala Glu 275 280 285 Gln Leu Lys Asn ThrGly Lys Val Gln Arg Gly Gln Leu Gly Val Ile 290 295 300 Ile Gln Glu ValSer Tyr Gly Leu Ala Gln Ser Phe Gly Leu Asp Lys 305 310 315 320 Ala GlyGly Ala Leu Ile Ala Lys Ile Leu Pro Gly Ser Pro Ala Glu 325 330 335 ArgAla Gly Leu Gln Ala Gly Asp Ile Val Leu Ser Leu Asp Gly Gly 340 345 350Glu Ile Arg Ser Ser Gly Asp Leu Pro Val Met Val Gly Ala Ile Thr 355 360365 Pro Gly Lys Glu Val Ser Leu Gly Val Trp Arg Lys Gly Glu Glu Ile 370375 380 Thr Ile Lys Val Lys Leu Gly Asn Ala Ala Glu His Ile Gly Ala Ser385 390 395 400 Ser Lys Thr Asp Glu Ala Pro Tyr Thr Glu Gln Gln Ser GlyThr Phe 405 410 415 Ser Val Glu Ser Ala Gly Ile Thr Leu Gln Thr His ThrAsp Ser Ser 420 425 430 Gly Gly His Leu Val Val Val Arg Val Ser Asp AlaAla Glu Arg Ala 435 440 445 Gly Leu Arg Arg Gly Asp Glu Ile Leu Ala ValGly Gln Val Pro Val 450 455 460 Asn Asp Glu Ala Gly Phe Arg Lys Ala MetAsp Lys Ala Gly Lys Asn 465 470 475 480 Val Pro Leu Leu Ile Met Arg ArgGly Asn Thr Leu Phe Ile Ala Leu 485 490 495 Asn Leu Gln 7 1110 DNABacteria 7 atgttcaaaa aataccaata cctcgctttg gcagcactgt gtgccgcctcgctggcaggc 60 tgcgacaagg caggcagctt tttcggtgcg gacaaaaaag aagcatccttcgtagaacgc 120 atcgaacaca ccaaagacga cggcagcgtc agtatgctgc tgcccgactttgcccaactg 180 gttcaaagtg aaggtccggc agtcgtcaat attcaggcag cccccgccccgcgcacccaa 240 aacggcagcg gcaatgccga aaacgattcc gacccgattg ccgacaacgacccgttctac 300 gaatttttca aacgcctcgt cccgaatatg cccgaaatcc cccaagaagaagcagatgac 360 ggcggattga acttcggttc gggcttcatc atcagcaaag acggctacatcctgaccaat 420 acccacgtcg ttaccggcat gggcagtatc aaagtcctgc tcaacgacaagcgcgaatat 480 accgccaaac tcatcggttc ggatgtccaa tccgatgtcg cccttctgaaaatcgacgca 540 acggaagagc tgcccgtcgt caaaatcggc aatcccaaag atttgaaaccgggcgaatgg 600 gtcgccgcca tcggcgcgcc cttcggcttc gacaacagcg tgaccgccggcatcgtgtcc 660 gccaaaggca gaagcctgcc caacgaaagc tacacaccct tcatccaaaccgacgttgcc 720 atcaatccgg gcaactccgg cggcccgctg ttcaacttaa aaggacaggtcgtcggcatc 780 aactcgcaaa tatacagccg cagcggcgga ttcatgggca tttccttcgccatcccgatt 840 gacgttgcca tgaatgtcgc cgaacagctg aaaaacaccg gcaaagtccaacgcggacaa 900 ctgggcgtga ttattcaaga agtatcctac ggtttggcac aatcgttcggtttggacaaa 960 gccggcggcg cactgattgc caaaatcctg cccggcagcc ccgcagaacgtgccggcctg 1020 caggcgggcg acatcgtcct cagcctcgac ggcggagaaa tacgttcttccggcgacctt 1080 cccgttatgg tcggcgccat tacgccggga 1110 8 370 PRT Bacteria8 Met Phe Lys Lys Tyr Gln Tyr Leu Ala Leu Ala Ala Leu Cys Ala Ala 1 5 1015 Ser Leu Ala Gly Cys Asp Lys Ala Gly Ser Phe Phe Gly Ala Asp Lys 20 2530 Lys Glu Ala Ser Phe Val Glu Arg Ile Glu His Thr Lys Asp Asp Gly 35 4045 Ser Val Ser Met Leu Leu Pro Asp Phe Ala Gln Leu Val Gln Ser Glu 50 5560 Gly Pro Ala Val Val Asn Ile Gln Ala Ala Pro Ala Pro Arg Thr Gln 65 7075 80 Asn Gly Ser Gly Asn Ala Glu Asn Asp Ser Asp Pro Ile Ala Asp Asn 8590 95 Asp Pro Phe Tyr Glu Phe Phe Lys Arg Leu Val Pro Asn Met Pro Glu100 105 110 Ile Pro Gln Glu Glu Ala Asp Asp Gly Gly Leu Asn Phe Gly SerGly 115 120 125 Phe Ile Ile Ser Lys Asp Gly Tyr Ile Leu Thr Asn Thr HisVal Val 130 135 140 Thr Gly Met Gly Ser Ile Lys Val Leu Leu Asn Asp LysArg Glu Tyr 145 150 155 160 Thr Ala Lys Leu Ile Gly Ser Asp Val Gln SerAsp Val Ala Leu Leu 165 170 175 Lys Ile Asp Ala Thr Glu Glu Leu Pro ValVal Lys Ile Gly Asn Pro 180 185 190 Lys Asp Leu Lys Pro Gly Glu Trp ValAla Ala Ile Gly Ala Pro Phe 195 200 205 Gly Phe Asp Asn Ser Val Thr AlaGly Ile Val Ser Ala Lys Gly Arg 210 215 220 Ser Leu Pro Asn Glu Ser TyrThr Pro Phe Ile Gln Thr Asp Val Ala 225 230 235 240 Ile Asn Pro Gly AsnSer Gly Gly Pro Leu Phe Asn Leu Lys Gly Gln 245 250 255 Val Val Gly IleAsn Ser Gln Ile Tyr Ser Arg Ser Gly Gly Phe Met 260 265 270 Gly Ile SerPhe Ala Ile Pro Ile Asp Val Ala Met Asn Val Ala Glu 275 280 285 Gln LeuLys Asn Thr Gly Lys Val Gln Arg Gly Gln Leu Gly Val Ile 290 295 300 IleGln Glu Val Ser Tyr Gly Leu Ala Gln Ser Phe Gly Leu Asp Lys 305 310 315320 Ala Gly Gly Ala Leu Ile Ala Lys Ile Leu Pro Gly Ser Pro Ala Glu 325330 335 Arg Ala Gly Leu Gln Ala Gly Asp Ile Val Leu Ser Leu Asp Gly Gly340 345 350 Glu Ile Arg Ser Ser Gly Asp Leu Pro Val Met Val Gly Ala IleThr 355 360 365 Pro Gly 370 9 390 DNA Bacteria 9 aaagaagtca gcctcggcgtatggcgcaaa ggcgaagaaa tcacaatcaa agtcaagctg 60 ggcaacgccg ccgagcatatcggcgcatca tccaaaacag atgaagcccc ctacaccgaa 120 cagcaatccg gtacgttctcggtcgaatcc gcaggcatta cccttcagac acataccgac 180 agcagcggcg gacacctcgtcgtcgtacgg gtttccgacg cggcagaacg cgcaggcttg 240 aggcgcggcg acgaaattcttgccgtcggg caagtccccg tcaatgacga agccggtttc 300 cgcaaagcta tggacaaggcaggcaaaaac gtccccctgc tgatcatgcg ccgtggcaac 360 acgctgttca tcgcattaaacctgcaataa 390 10 129 PRT Bacteria 10 Lys Glu Val Ser Leu Gly Val TrpArg Lys Gly Glu Glu Ile Thr Ile 1 5 10 15 Lys Val Lys Leu Gly Asn AlaAla Glu His Ile Gly Ala Ser Ser Lys 20 25 30 Thr Asp Glu Ala Pro Tyr ThrGlu Gln Gln Ser Gly Thr Phe Ser Val 35 40 45 Glu Ser Ala Gly Ile Thr LeuGln Thr His Thr Asp Ser Ser Gly Gly 50 55 60 His Leu Val Val Val Arg ValSer Asp Ala Ala Glu Arg Ala Gly Leu 65 70 75 80 Arg Arg Gly Asp Glu IleLeu Ala Val Gly Gln Val Pro Val Asn Asp 85 90 95 Glu Ala Gly Phe Arg LysAla Met Asp Lys Ala Gly Lys Asn Val Pro 100 105 110 Leu Leu Ile Met ArgArg Gly Asn Thr Leu Phe Ile Ala Leu Asn Leu 115 120 125 Gln 11 32 DNAArtificial Sequence Primer 11 ggaattccat atgttcaaaa aataccaata cc 32 1230 DNA Artificial Sequence Primer 12 cgccgctcga gttgcaggtt taatgcgatg 3013 33 DNA Artificial Sequence Primer 13 gatatacata tgttcaaaaa ataccaatacctc 33 14 33 DNA Artificial Sequence Primer 14 ctagggctcg agtcccggcgtaatggcgcc gac 33 15 33 DNA Artificial Sequence Primer 15 gatatacatatgaaagaagt cagcctcggc gta 33 16 33 DNA Artificial Sequence Primer 16ctagggctcg agttgcaggt ttaatgcgat gaa 33

What is claimed is:
 1. An isolated polynucleotide comprising a firstpolynucleotide sequence or the full complement of the firstpolynucleotide sequence, wherein the first polynucleotide sequenceencodes a polypeptide selected from the group consisting of SEQ ID NO:2,4 or
 6. 2. The isolated polynucleotide of claim 1, wherein the isolatedpolynucleotide comprises the first polynucleotide sequence.
 3. Theisolated polynucleotide of claim 2, wherein the first polynucleotidesequence encodes the polypeptide consisting of SEQ ID NO:2.
 4. Theisolated polynucleotide of claim 3, wherein the isolated polynucleotideconsists of the first polynucleotide sequence.
 5. The isolatedpolynucleotide of claim 2, wherein the first polynucleotide sequenceencodes the polypeptide consisting of SEQ ID NO:4.
 6. The isolatedpolynucleotide of claim 5, wherein the isolated polynucleotide consistsof the first polynucleotide sequence.
 7. The isolated polynucleotide ofclaim 2, wherein the first polynucleotide sequence encodes thepolypeptide consisting of SEQ ID NO:6.
 8. The isolated polynucleotide ofclaim 7, wherein the isolated polynucleotide consists of the firstpolynucleotide sequence.
 9. An expression vector comprising the isolatedpolynucleotide of claim
 1. 10. A host cell comprising the expressionvector of claim
 9. 11. An immunogenic composition comprising theisolated polynucleotide of claim 1 and a pharmaceutically acceptablecarrier.
 12. The immunogenic composition of claim 11, further comprisingan adjuvant.
 13. An isolated polynucleotide comprising a firstpolynucleotide or the full complement of the first polynucleotidesequence, wherein the first polynucleotide sequence is selected from thegroup consisting of SEQ ID NO: 1, 3 or
 5. 14. The isolatedpolynucleotide of claim 13, wherein the isolated polynucleotidecomprises the first polynucleotide sequence.
 15. The isolatedpolynucleotide of claim 14, wherein the first polynucleotide sequenceconsists of SEQ ID NO:
 1. 16. The isolated polynucleotide of claim 15,wherein the isolated polynucleotide consists of the first polynucleotidesequence.
 17. The isolated polynucleotide of claim 14, wherein the firstpolynucleotide sequence consists of SEQ ID NO:3.
 18. The isolatedpolynucleotide of claim 17, wherein the isolated polynucleotide consistsof the first polynucleotide sequence.
 19. The isolated polynucleotide ofclaim 14, wherein the first polynucleotide sequence consists of SEQ IDNO:5.
 20. The isolated polynucleotide of claim 19, wherein the isolatedpolynucleotide consists of the first polynucleotide sequence.
 21. Anexpression vector comprising the isolated polynucleotide of claim 13.22. A host cell comprising the expression vector of claim
 21. 23. Animmunogenic composition comprising the isolated polynucleotide of claim13 and a pharmaceutically acceptable carrier.
 24. The immunogeniccomposition of claim 23, further comprising an adjuvant.